Polypeptides Having Xylanase Activity And Polynucleotides Encoding Same

ABSTRACT

The present invention relates to isolated polypeptides having xylanase activity, catalytic domains, carbohydrate binding modules and encoding the polypeptides, catalytic domains or carbohydrate binding modules. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or carbohydrate binding modules.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to polypeptides having xylanase activity,catalytic domains, and carbohydrate binding modules, and polynucleotidesencoding the polypeptides, catalytic domains, and carbohydrate bindingmodules. The invention also relates to nucleic acid constructs, vectors,and host cells comprising the polynucleotides as well as methods ofproducing and using the polypeptides, catalytic domains, andcarbohydrate binding modules.

2. Description of the Related Art

Lignocellulose, the world's largest renewable biomass resource, iscomposed mainly of lignin, cellulose, and hemicellulose, of which alarge part of the latter is xylan. Xylanases (e.g.,endo-1,4-beta-xylanase, EC 3.2.1.8) hydrolyze internal β-1,4-xylosidiclinkages in xylan to produce smaller molecular weight xylose andxylo-oligomers. Xylans are polysaccharides formed from1,4-β-glycoside-linked D-xylopyranoses.

Cellulose is a polymer of the simple glucose covalently linked bybeta-1,4-bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose. Once the cellulose is converted toglucose, the glucose is easily fermented by yeast into ethanol.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the cellulose is converted to glucose,the glucose is easily fermented by yeast into ethanol.

Yoshioka et al., 1981, Agric. Biol. Chem. 45(3): 579-586, discloseproduction and characterization of a thermostable xylanase fromTalaromyces byssochiamydoides YH-50. Yoshioka et al., 1981, Agric. Biol.Chem. 45(11): 2425-2432, disclose purification and properties of athermostable xylanase from Talaromyces byssochlamydoides YH-50.Hayashida et al., 1988, Methods In Enzymology 160: 675-678, disclose aTalaromyces byssochlamydoides xylanase. WO 02/24926 discloses aTalaromyces emersonii GH10 xylanase (GENESEQP:AAU99346).

There is a need in the art to improve cellulolytic enzyme compositionsthrough supplementation with additional enzymes to increase efficiencyand to provide cost-effective enzyme solutions for degradation oflignocellulose.

The present invention provides polypeptides having xylanase activity andpolynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides having xylanaseactivity selected from the group consisting of:

(a) a polypeptide having at least 90% sequence identity to the maturepolypeptide of SEQ ID NO: 2, or to the mature polypeptide of SEQ ID NO:6;

(b) a polypeptide encoded by a polynucleotide having at least 90%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the cDNA sequence thereof, or to the mature polypeptide codingsequence of SEQ ID NO: 5 or the cDNA sequence thereof;

(c) a variant of the mature polypeptide of SEQ ID NO: 2, or of themature polypeptide of SEQ ID NO: 6 comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions; and

(d) a fragment of the polypeptide of (a), (b), or (c) that has xylanaseactivity.

The present invention also relates to isolated polypeptides comprising acatalytic domain selected from the group consisting of:

(a) a catalytic domain having at least 90% sequence identity to aminoacids 24 to 340 of SEQ ID NO: 2 or to amino acids 24 to 341 of SEQ IDNO: 6;

(b) a catalytic domain encoded by a polynucleotide having at least 90%sequence identity to nucleotides 157 to 1339 of SEQ ID NO: 1 or the cDNAsequence thereof, or having at least 90% sequence identity tonucleotides 151 to 1387 of SEQ ID NO: 5 or the cDNA sequence thereof;

(c) a variant of amino acids 24 to 340 of SEQ ID NO: 2, or of acids 24to 341 of SEQ ID NO: 6 comprising a substitution, deletion, and/orinsertion at one or more (e.g., several) positions; and

(d) a fragment of the catalytic domain of (a), (b), or (c) that hasxylanase activity.

The present invention also relates to isolated polypeptides comprising acarbohydrate binding module selected from the group consisting of:

(a) a carbohydrate binding module having at least 90% sequence identityto amino acids 373 to 406 of SEQ ID NO: 2 or having at least 90%sequence identity to amino acids 371 to 406 of SEQ ID NO: 6;

(b) a carbohydrate binding module encoded by a polynucleotide having atleast 90% sequence identity to nucleotides 1436 to 1537 of SEQ ID NO: 1or the cDNA sequence thereof, or encoded by a polynucleotide having atleast 90% sequence identity to nucleotides 1480-1587 of SEQ ID NO: 5;

(c) a variant of amino acids 373 to 406 of SEQ ID NO: 2, or of aminoacids 371 to 406 of SEQ ID NO: 6, comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions; and

(d) a fragment of the carbohydrate binding module of (a), (b), or (c)that has binding activity.

The present invention also relates to isolated polynucleotides encodingthe polypeptides of the present invention; nucleic acid constructs,recombinant expression vectors, and recombinant host cells comprisingthe polynucleotides; and methods of producing the polypeptides.

The present invention also relates to processes for degrading acellulosic or xylan-containing material, comprising: treating thecellulosic or xylan-containing material with an enzyme composition inthe presence of a polypeptide having xylanase activity of the presentinvention. In one aspect, the processes further comprise recovering thedegraded cellulosic or xylan-containing material.

The present invention also relates to processes of producing afermentation product, comprising: (a) saccharifying a cellulosic orxylan-containing material with an enzyme composition in the presence ofa polypeptide having xylanase activity of the present invention; (b)fermenting the saccharified cellulosic or xylan-containing material withone or more (e.g., several) fermenting microorganisms to produce thefermentation product; and (c) recovering the fermentation product fromthe fermentation.

The present invention also relates to processes of fermenting acellulosic or xylan-containing material, comprising: fermenting thecellulosic or xylan-containing material with one or more (e.g., several)fermenting microorganisms, wherein the cellulosic or xylan-containingmaterial is saccharified with an enzyme composition in the presence of apolypeptide having xylanase activity of the present invention. In oneaspect, the fermenting of the cellulosic or xylan-containing materialproduces a fermentation product. In another aspect, the processesfurther comprise recovering the fermentation product from thefermentation.

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to23 of SEQ ID NO: 2, or a signal peptide comprising or consisting ofamino acids 1 to 23 of SEQ ID NO: 6, which is operably linked to a geneencoding a protein, wherein the protein is foreign to the signalpeptide; nucleic acid constructs, expression vectors, and recombinanthost cells comprising the polynucleotides; and methods of producing aprotein.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 shows hydrolysis results of washed ground-sieved alkalinepretreated corn cobs (GS-APCC) as a substrate at pH 4.0 from 50° C. to65° C. by the Rasamsonia byssochlamydoides GH10 xylanase (P24GTR)supplemented with Talaromyces emersonii GH3 beta-xylosidase.

DEFINITIONS

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. Acetylxylan esteraseactivity is preferably determined using 0.5 mM p-nitrophenylacetate assubstrate in 50 mM sodium acetate pH 5.0 containing 0.01% TWEEN™ 20(polyoxyethylene sorbitan monolaurate). One unit of acetylxylan esteraseis defined as the amount of enzyme capable of releasing 1 μmole ofp-nitrophenolate anion per minute at pH 5, 25° C.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase.Alpha-L-arabinofuranosidase activity is preferably determined using 5 mgof medium viscosity wheat arabinoxylan (Megazyme International Ireland,Ltd., Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5in a total volume of 200 μl for 30 minutes at 40° C. followed byarabinose analysis by AMINEX® HPX-87H column chromatography (Bio-RadLaboratories, Inc., Hercules, Calif., USA).

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. Alpha-glucuronidase activity is preferably determined accordingto de Vries, 1998, J. Bacteriol. 180: 243-249. One unit ofalpha-glucuronidase equals the amount of enzyme capable of releasing 1μmole of glucuronic or 4-O-methylglucuronic acid per minute at pH 5, 40°C.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.Beta-glucosidase activity is preferably determined usingp-nitrophenyl-beta-D-glucopyranoside as substrate according to theprocedure of Venturi et al., 2002, J. Basic Microbiol. 42: 55-66. Oneunit of beta-glucosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 25° C., pH 4.8 from 1 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodiumcitrate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides to remove successive D-xylose residuesfrom non-reducing termini. Beta-xylosidase activity is preferablydetermined using 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100mM sodium citrate containing 0.01% TWEEN® 20 at pH 5, 40° C. One unit ofbeta-xylosidase is defined as 1.0 μmole of p-nitrophenolate anionproduced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside in 100 mM sodium citrate containing 0.01%TWEEN® 20.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Carbohydrate binding module: The term “carbohydrate binding module”means the region within a carbohydrate-active enzyme that providescarbohydrate-binding activity (Boraston et al., 2004, Biochem. J. 383:769-781). A majority of known carbohydrate binding modules (CBMs) arecontiguous amino acid sequences with a discrete fold. The carbohydratebinding module (CBM) is typically found either at the N-terminal or atthe C-terminal extremity of an enzyme. Some CBMs are known to havespecificity for cellulose.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176)that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages incellulose, cellooligosaccharides, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the reducing end(cellobiohydrolase I) or non-reducing end (cellobiohydrolase II) of thechain (Teeri, 1997, Trends in Biotechnology 15: 160-167; Teeri et al.,1998, Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity ispreferably determined according to the procedures described by Lever etal., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBSLetters 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic enzyme activity include:(1) measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., 2006, BiotechnologyAdvances 24: 452-481. Total cellulolytic activity is usually measuredusing insoluble substrates, including Whatman No 1 filter paper,microcrystalline cellulose, bacterial cellulose, algal cellulose,cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No 1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity ispreferably determined by measuring the increase in hydrolysis of acellulosic material by cellulolytic enzyme(s) under the followingconditions: 1-50 mg of cellulolytic enzyme protein/g of cellulose in PCS(or other pretreated cellulosic material) for 3-7 days at a suitabletemperature, such as 40° C.-80° C., e.g., 50° C., 55° C., 60° C., 65°C., or 70° C., and a suitable pH, such as 4-9, e.g., 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, or 8.5, compared to a control hydrolysis withoutaddition of cellulolytic enzyme protein. Typical conditions are 1 mlreactions, washed or unwashed PCS, 5% insoluble solids, 50 mM sodiumacetate pH 5, 1 mM MnSO₄, 50° C., 55° C., or 60° C., 72 hours, sugaranalysis by AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA).

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, and wood (including forestryresidue) (see, for example, Wiselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd,1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier etal., 1999, Recent Progress in Bioconversion of Lignocellulosics, inAdvances in Biochemical Engineering/Biotechnology, T. Scheper, managingeditor, Volume 65, pp. 23-40, Springer-Verlag, New York). It isunderstood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is any biomass material. In another preferredaspect, the cellulosic material is lignocellulose, which comprisescellulose, hemicelluloses, and lignin.

In one aspect, the cellulosic material is agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, or wood (including forestryresidue).

In another aspect, the cellulosic material is arundo, bagasse, bamboo,corn cob, corn fiber, corn stover, miscanthus, rice straw, switchgrass,or wheat straw.

In another aspect, the cellulosic material is aspen, eucalyptus, fir,pine, poplar, spruce, or willow.

In another aspect, the cellulosic material is algal cellulose, bacterialcellulose, cotton linter, filter paper, microcrystalline cellulose(e.g., AVICEL®), or phosphoric-acid treated cellulose.

In another aspect, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Endoglucanase: The term “endoglucanase” means anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such ascereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). Endoglucanase activity ispreferably determined using carboxymethyl cellulose (CMC) as substrateaccording to the procedure of Ghose, 1987, Pure and Appl. Chem. 59:257-268, at pH 5, 40° C.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Family 10 glycoside hydrolase: The term “Family 10 glycoside hydrolase”or “Family GH10” or “GH10” means a polypeptide falling into theglycoside hydrolase Family 10 according to Henrissat, 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat and Bairoch, 1996,Updating the sequence-based classification of glycosyl hydrolases,Biochem. J. 316: 695-696.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat, 1991, Biochem. J.280: 309-316, and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.The enzymes in this family were originally classified as a glycosidehydrolase family based on measurement of very weakendo-1,4-beta-D-glucanase activity in one family member. The GH61s haverecently been classified as lytic polysaccharide monooxygenases (Quinlanet al., 2011, Proc. Natl. Acad. Sci. USA 208: 15079-15084; Phillips etal., 2011, ACS Chem. Biol. 6: 1399-1406; Lin et al., 2012, Structure 20:1051-1061).

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)groups from esterified sugar, which is usually arabinose in naturalbiomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate).Feruloyl esterase is also known as ferulic acid esterase,hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA,cinnAE, FAE-I, or FAE-II. Feruloyl esterase activity is preferablydetermined using 0.5 mM p-nitrophenylferulate as substrate in 50 mMsodium acetate pH 5.0. One unit of feruloyl esterase equals the amountof enzyme capable of releasing 1 μmole of p-nitrophenolate anion perminute at pH 5, 25° C.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide main; wherein the fragment has xylanaseactivity. In one aspect, a fragment contains at least 320 amino acidresidues, e.g., at least 340 amino acid residues or at least 360 aminoacid residues of SEQ ID NO: 2 or of SEQ ID NO: 6.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom andShoham, 2003, Current Opinion In Microbiology 6(3): 219-228).Hemicellulases are key components in the degradation of plant biomass.Examples of hemicellulases include, but are not limited to, anacetylmannan esterase, an acetylxylan esterase, an arabinanase, anarabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, agalactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, amannosidase, a xylanase, and a xylosidase. The substrates for theseenzymes, hemicelluloses, are a heterogeneous group of branched andlinear polysaccharides that are bound via hydrogen bonds to thecellulose microfibrils in the plant cell wall, crosslinking them into arobust network. Hemicelluloses are also covalently attached to lignin,forming together with cellulose a highly complex structure. The variablestructure and organization of hemicelluloses require the concertedaction of many enzymes for its complete degradation. The catalyticmodules of hemicellulases are either glycoside hydrolases (GHs) thathydrolyze glycosidic bonds, or carbohydrate esterases (CEs), whichhydrolyze ester linkages of acetate or ferulic acid side groups. Thesecatalytic modules, based on homology of their primary sequence, can beassigned into GH and CE families. Some families, with an overall similarfold, can be further grouped into clans, marked alphabetically (e.g.,GH-A). A most informative and updated classification of these and othercarbohydrate active enzymes is available in the Carbohydrate-ActiveEnzymes (CAZy) database. Hemicellulolytic enzyme activities can bemeasured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59:1739-1752, at a suitable temperature, e.g., 50° C., 55° C., or 60° C.,and pH, e.g., 4.5, 5.0 or 5.5.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 0.2×SSC, 0.2% SDSat 65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., recombinantproduction in a host cell; multiple copies of a gene encoding thesubstance; and use of a stronger promoter than the promoter naturallyassociated with the gene encoding the substance).

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 0.2×SSC, 0.2% SDSat 50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 24 to 406 of SEQ ID NO: 2 (P24GTR) based onthe SignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6)that predicts amino acids 1 to 23 of SEQ ID NO: 2 are a signal peptide.In another aspect, the mature polypeptide is amino acids 24 to 406 ofSEQ ID NO: 6 (P34RRZ) based on the SignalP program (Nielsen et al.,1997, Protein Engineering 10: 1-6) that predicts amino acids 1 to 23 ofSEQ ID NO: 6 are a signal peptide. It is known in the art that a hostcell may produce a mixture of two of more different mature polypeptides(i.e., with a different C-terminal and/or N-terminal amino acid)expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving xylanase activity. In one aspect, the mature polypeptide codingsequence is nucleotides 157 to 1537 of SEQ ID NO: 1 (D82RVA) or the cDNAsequence thereof based on the SignalP program (Nielsen et al., 1997,supra) that predicts nucleotides 1 to 156 of SEQ ID NO: 1 encode asignal peptide. In another aspect, the mature polypeptide codingsequence is nucleotides 151 to 1587 of SEQ ID NO: 5 (D24EPN) or the cDNAsequence thereof based on the SignalP program (Nielsen et al., 1997,supra) that predicts nucleotides 1 to 150 of SEQ ID NO: 5 encode asignal peptide.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide that catalyzes the enhancement of the hydrolysis of acellulosic material by enzyme having cellulolytic activity. Cellulolyticenhancing activity is preferably determined by measuring the increase inreducing sugars or the increase of the total of cellobiose and glucosefrom the hydrolysis of a cellulosic material by cellulolytic enzymeunder the following conditions: 1-50 mg of total protein/g of cellulosein pretreated corn stover (PCS), wherein total protein is comprised of50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/w protein of aGH61 polypeptide for 1-7 days at a suitable temperature, such as 40°C.-80° C., e.g., 50° C., 55° C., 60° C., 65° C., or 70° C., and asuitable pH, such as 4-9, e.g., 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0,or 8.5, compared to a control hydrolysis with equal total proteinloading without cellulolytic enhancing activity (1-50 mg of cellulolyticprotein/g of cellulose in PCS).

In one aspect, GH61 polypeptide enhancing activity is determined using amixture of CELLUCLAST® 1.5 L (Novozymes NS, Bagsværd, Denmark) in thepresence of 2-3% of total protein weight Aspergillus oryzaebeta-glucosidase (recombinantly produced in Aspergillus oryzae accordingto WO 02/095014) or 2-3% of total protein weight Aspergillus fumigatusbeta-glucosidase (recombinantly produced in Aspergillus oryzae asdescribed in WO 02/095014) of cellulase protein loading is used as thesource of the cellulolytic activity.

In another aspect, GH61 polypeptide enhancing activity is determinedaccording to WO 2013/028928 for high temperature compositions.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid, alkaline pretreatment, neutral pretreatment,or any pretreatment known in the art.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 3.0.0, 5.0.0 or later. The parametersused are gap open penalty of 10, gap extension penalty of 0.5, and theEBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the -nobriefoption) is used as the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having xylanase activity. In one aspect, a subsequence containsat least 960 nucleotides, e.g., at least 1020 nucleotides or at least1080 nucleotides of SEQ ID NO: 1 or of SEQ ID NO: 5.

Variant: The term “variant” means a polypeptide having xylanase activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion, at one or more (e.g., several) positions. A substitution meansreplacement of the amino acid occupying a position with a differentamino acid; a deletion means removal of the amino acid occupying aposition; and an insertion means adding an amino acid adjacent to andimmediately following the amino acid occupying a position.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 45° C.

Xylan-containing material: The term “xylan-containing material” meansany material comprising a plant cell wall polysaccharide containing abackbone of beta-(1-4)-linked xylose residues. Xylans of terrestrialplants are heteropolymers possessing a beta-(1-4)-D-xylopyranosebackbone, which is branched by short carbohydrate chains. They compriseD-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or variousoligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose,and D-glucose. Xylan-type polysaccharides can be divided into homoxylansand heteroxylans, which include glucuronoxylans,(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, andcomplex heteroxylans. See, for example, Ebringerova et al., 2005, Adv.Polym. Sci. 186: 1-67.

In the processes of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, 2006, Journal of the Science of Food and Agriculture 86(11):1636-1647; Spanikova and Biely, 2006, FEBS Letters 580(19): 4597-4601;Herrmann et al., 1997, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. The most common total xylanolytic activity assay is basedon production of reducing sugars from polymeric 4-O-methylglucuronoxylan as described in Bailey et al., 1992, Interlaboratorytesting of methods for assay of xylanase activity, Journal ofBiotechnology 23(3): 257-270. Xylanase activity can also be determinedwith 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100(4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200 mMsodium phosphate pH 6 at 37° C. One unit of xylanase activity is definedas 1.0 μmole of azurine produced per minute at 37° C., pH 6 from 0.2%AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6.

Xylan degrading activity is preferably determined by measuring theincrease in hydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St.Louis, Mo., USA) by xylan-degrading enzyme(s) under the followingtypical conditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5mg of xylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50°C., 24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide(PHBAH) assay as described by Lever, 1972, Anal. Biochem. 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. Xylanase activity is preferably determined with 0.2%AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodiumphosphate pH 6 at 37° C. One unit of xylanase activity is defined as 1.0μmole of azurine produced per minute at 37° C., pH 6 from 0.2%AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6.

The polypeptides of the present invention have at least 20%, e.g., atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, and at least 100% of the xylanase activity ofthe mature polypeptide of SEQ ID NO: 2 or of the mature polypeptide ofSEQ ID NO: 6.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having XylanaseActivity

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 60%, e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%,which have xylanase activity. In one aspect, the polypeptides differ byup to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from themature polypeptide of SEQ ID NO: 2.

In another embodiment, the present invention relates to isolatedpolypeptides having a sequence identity to the mature polypeptide of SEQID NO: 6 of at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%, which have xylanase activity. In one aspect, thepolypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, or 10, from the mature polypeptide of SEQ ID NO: 6.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6, or anallelic variant thereof; or is a fragment thereof having xylanaseactivity. In another aspect, the polypeptide comprises or consists ofthe mature polypeptide of SEQ ID NO: 2. In another aspect, thepolypeptide comprises or consists of amino acids 24 to 406 of SEQ ID NO:2. In another aspect, the polypeptide comprises or consists of aminoacids 24 to 406 of SEQ ID NO: 6.

In another embodiment, the present invention relates to an isolatedpolypeptide having xylanase activity encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1 or of SEQ ID NO: 5, (ii) the cDNA sequence of SEQ ID NO: 1 or of SEQID NO: 5, or (iii) the full-length complement of (i) or (ii) (Sambrooket al., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, ColdSpring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 5, or a subsequencethereof, as well as the polypeptide of SEQ ID NO: 2 or of SEQ ID NO: 6,the mature polypeptide thereof, or a fragment thereof, may be used todesign nucleic acid probes to identify and clone DNA encodingpolypeptides having xylanase activity from strains of different generaor species according to methods well known in the art. In particular,such probes can be used for hybridization with the genomic DNA or cDNAof a cell of interest, following standard Southern blotting procedures,in order to identify and isolate the corresponding gene therein. Suchprobes can be considerably shorter than the entire sequence, but shouldbe at least 15, e.g., at least 25, at least 35, or at least 70nucleotides in length. Preferably, the nucleic acid probe is at least100 nucleotides in length, e.g., at least 200 nucleotides, at least 300nucleotides, at least 400 nucleotides, at least 500 nucleotides, atleast 600 nucleotides, at least 700 nucleotides, at least 800nucleotides, or at least 900 nucleotides in length. Both DNA and RNAprobes can be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having xylanase activity. Genomic or other DNAfrom such other strains may be separated by agarose or polyacrylamidegel electrophoresis, or other separation techniques. DNA from thelibraries or the separated DNA may be transferred to a suitable carriermaterials, such as nitrocellulose. In order to identify a clone or DNAthat hybridizes with SEQ ID NO: 1 or SEQ ID NO: 5, the maturepolypeptide coding sequences thereof, or a subsequence thereof, thecarrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1 or SEQ ID NO: 5; (ii) the mature polypeptide codingsequence of SEQ ID NO: 1 or of SEQ ID NO: 5; (iii) the cDNA sequence ofSEQ ID NO: 1 or of SEQ ID NO: 5, or the mature polypeptide codingsequence thereof; (iv) the full-length complement thereof; or (v) asubsequence thereof; under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using, for example, X-ray film or any otherdetection means known in the art.

In one aspect, the nucleic acid probe is a polynucleotide that encodesthe polypeptide of SEQ ID NO: 2; the mature polypeptide thereof; or afragment thereof. In another aspect, the nucleic acid probe is SEQ IDNO: 1; the mature polypeptide coding sequence thereof; or the cDNAsequence thereof. In another aspect, the nucleic acid probe is thepolynucleotide contained in Rasamsonia byssochiamydoides CBS 413.71,wherein the polynucleotide encodes a polypeptide having xylanaseactivity. In another aspect, the nucleic acid probe is the maturepolypeptide coding sequence contained in Rasamsonia byssochiamydoidesCBS 413.71.

In one aspect, the nucleic acid probe is a polynucleotide that encodesthe polypeptide of SEQ ID NO: 6; the mature polypeptide thereof; or afragment thereof. In another aspect, the nucleic acid probe is SEQ IDNO: 5; the mature polypeptide coding sequence thereof; or the cDNAsequence thereof. In another aspect, the nucleic acid probe is thepolynucleotide contained in Rasamsonia byssochlamydoides CBS 150.75,wherein the polynucleotide encodes a polypeptide having xylanaseactivity. In another aspect, the nucleic acid probe is the maturepolypeptide coding sequence contained in Rasamsonia byssochlamydoidesCBS 150.75.

In another embodiment, the present invention relates to an isolatedpolypeptide having xylanase activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the cDNA sequence thereof of at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%.

In another embodiment, the present invention relates to an isolatedpolypeptide having xylanase activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 5 or the cDNA sequence thereof of at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 2 comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions. In oneaspect, the number of amino acid substitutions, deletions and/orinsertions introduced into the mature polypeptide of SEQ ID NO: 2 is upto 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 6 comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions. In oneaspect, the number of amino acid substitutions, deletions and/orinsertions introduced into the mature polypeptide of SEQ ID NO: 6 is upto 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for xylanase activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

In each of the embodiments above, in one aspect, an isolated polypeptidehaving xylanase activity of the present invention has at least 10%,e.g., at least 15% and at least 20%, more xylanase activity at pH 4.0and 60° C. or 65° C. than at pH 4.0 and 50° C. In one aspect, xylanaseactivity is preferably determined using washed ground-sieved alkalinepretreated corn cobs (GS-APCC) as a substrate according to the protocoldescribed in Example 7 for 72 hours in 50 mM sodium acetate (pH 4.0 to5.5) or 50 mM Tris (pH 6.0 to 7.0) buffer containing 1 mM manganesesulfate.

In each of the embodiments above, in another aspect, an isolatedpolypeptide having xylanase activity of the present invention has atleast 10%, e.g., at least 15% and at least 20%, more xylanase activityat pH 4.0 and 50° C., 55° C., 60° C., or 65° C. than at pH 6.0 and 50°C., 55° C., 60° C., or 65° C., respectively. In one aspect, xylanaseactivity is preferably determined using washed ground-sieved alkalinepretreated corn cobs (GS-APCC) as a substrate according to the protocoldescribed in Example 7 for 72 hours in 50 mM sodium acetate (pH 4.0 to5.5) or 50 mM Tris (pH 6.0 to 7.0) buffer containing 1 mM manganesesulfate.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Xylanase Activity

A polypeptide having xylanase activity of the present invention may beobtained from microorganisms of any genus. For purposes of the presentinvention, the term “obtained from” as used herein in connection with agiven source shall mean that the polypeptide encoded by a polynucleotideis produced by the source or by a strain in which the polynucleotidefrom the source has been inserted. In one aspect, the polypeptideobtained from a given source is secreted extracellularly.

The polypeptide may be a fungal polypeptide. In one aspect, thepolypeptide is a Rasamsonia polypeptide. In another aspect, thepolypeptide is a Rasamsonia byssochlamydoides polypeptide. In anotheraspect, the polypeptide is a Rasamsonia byssochlamydoides CBS 413.71polypeptide. In another aspect, the polypeptide is a Rasamsoniabyssochlamydoides CBS 150.75 polypeptide.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents. For example, the species Rasamsoniabyssochlamydoides is sometimes referred to as Talaromycesbyssochlamydoides, or by its anamorph Paecilomyces byssochlamydoides.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Catalytic Domains

In one embodiment, the present invention also relates to catalyticdomains having a sequence identity to amino acids 24 to 340 of SEQ IDNO: 2 of at least 60%, e.g., at least 65%, at least 70%, at least 75%,at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%. In one aspect, the catalytic domains comprise amino acid sequencesthat differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or10, from amino acids 24 to 340 of SEQ ID NO: 2. The catalytic domainpreferably comprises or consists of amino acids 24 to 340 of SEQ ID NO:2 or an allelic variant thereof; or is a fragment thereof havingxylanase activity.

In another embodiment, the present invention also relates to catalyticdomains having a sequence identity to amino acids 24 to 341 of SEQ IDNO: 6 of at least 60%, e.g., at least 65%, at least 70%, at least 75%,at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%. In one aspect, the catalytic domains comprise amino acid sequencesthat differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or10, from amino acids 24 to 341 of SEQ ID NO: 6. The catalytic domainpreferably comprises or consists of amino acids 24 to 341 of SEQ ID NO:6 or an allelic variant thereof; or is a fragment thereof havingxylanase activity.

In another embodiment, the present invention also relates to catalyticdomains encoded by polynucleotides having a sequence identity tonucleotides 157 to 1339 of SEQ ID NO: 1 or the cDNA sequence thereof ofat least 60%, e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.The polynucleotide encoding the catalytic domain preferably comprises orconsists of nucleotides 157 to 1339 of SEQ ID NO: 1 or is the sequencecontained in Rasamsonia byssochiamydoides strain CBS 413.71.

In another embodiment, the present invention also relates to catalyticdomains encoded by polynucleotides having a sequence identity tonucleotides 151 to 1387 of SEQ ID NO: 5 or the cDNA sequence thereof ofat least 60%, e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.The polynucleotide encoding the catalytic domain preferably comprises orconsists of nucleotides 151 to 1387 of SEQ ID NO: 5 or is the sequencecontained in Rasamsonia byssochiamydoides strain CBS 150.75.

In another embodiment, the present invention also relates to catalyticdomain variants of amino acids 24 to 340 of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions. In one aspect, the number of amino acid substitutions,deletions and/or insertions introduced into the sequence of amino acids24 to 340 of SEQ ID NO: 2 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 8, 9, or10.

In another embodiment, the present invention also relates to catalyticdomain variants of amino acids 24 to 341 of SEQ ID NO: 6 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions. In one aspect, the number of amino acid substitutions,deletions and/or insertions introduced into the sequence of amino acids24 to 341 of SEQ ID NO: 6 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 8, 9, or10.

Carbohydrate Binding Modules

In one embodiment, the present invention also relates to carbohydratebinding modules having a sequence identity to amino acids 373 to 406 ofSEQ ID NO: 2 of at least 90%, e.g., at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%. In one aspect, the carbohydrate bindingmodules comprise amino acid sequences that differ by up to 10 aminoacids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from amino acids 373 to406 of SEQ ID NO: 2. The carbohydrate binding module preferablycomprises or consists of amino acids 373 to 406 of SEQ ID NO: 2 or anallelic variant thereof; or is a fragment thereof having carbohydratebinding activity.

In one embodiment, the present invention also relates to carbohydratebinding modules having a sequence identity to amino acids 371 to 406 ofSEQ ID NO: 6 of at least 90%, e.g., at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%. In one aspect, the carbohydrate bindingmodules comprise amino acid sequences that differ by up to 10 aminoacids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from amino acids 371 to406 of SEQ ID NO: 6. The carbohydrate binding module preferablycomprises or consists of amino acids 371 to 406 of SEQ ID NO: 6 or anallelic variant thereof; or is a fragment thereof having carbohydratebinding activity.

In another embodiment, the present invention also relates tocarbohydrate binding modules encoded by polynucleotides having asequence identity to nucleotides 1436 to 1537 of SEQ ID NO: 1 of atleast 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%. The polynucleotide encoding the carbohydrate binding modulepreferably comprises or consists of nucleotides 1436 to 1537 of SEQ IDNO: 1 or is the sequence contained in Rasamsonia byssochlamydoides CBS413.71.

In another embodiment, the present invention also relates tocarbohydrate binding modules encoded by polynucleotides having asequence identity to nucleotides 1480 to 1587 of SEQ ID NO: 5 of atleast 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%. The polynucleotide encoding the carbohydrate binding modulepreferably comprises or consists of nucleotides 1480 to 1587 of SEQ IDNO: 5 or is the sequence contained in Rasamsonia byssochlamydoides CBS150.75.

In another embodiment, the present invention also relates tocarbohydrate binding module variants of amino acids 373 to 406 of SEQ IDNO: 2 comprising a substitution, deletion, and/or insertion at one ormore (e.g., several) positions. In one aspect, the number of amino acidsubstitutions, deletions and/or insertions introduced into the sequenceof amino acids 373 to 406 of SEQ ID NO: 2 is up to 10, e.g., 1, 2, 3, 4,5, 6, 8, 9, or 10.

In another embodiment, the present invention also relates tocarbohydrate binding module variants of amino acids 371 to 406 of SEQ IDNO: 6 comprising a substitution, deletion, and/or insertion at one ormore (e.g., several) positions. In one aspect, the number of amino acidsubstitutions, deletions and/or insertions introduced into the sequenceof amino acids 371 to 406 of SEQ ID NO: 6 is up to 10, e.g., 1, 2, 3, 4,5, 6, 8, 9, or 10.

A catalytic domain operably linked to the carbohydrate binding modulemay be from a hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase, e.g., an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase,lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase, xylanase, or beta-xylosidase. The polynucleotideencoding the catalytic domain may be obtained from any prokaryotic,eukaryotic, or other source.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga polypeptide, a catalytic domain, or a carbohydrate binding module ofthe present invention, as described herein.

The techniques used to isolate or clone a polynucleotide are known inthe art and include isolation from genomic DNA or cDNA, or a combinationthereof. The cloning of the polynucleotides from genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligation activated transcription (LAT) andpolynucleotide-based amplification (NASBA) may be used. Thepolynucleotides may be cloned from a strain of Corynascus, or a relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the polynucleotide.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for synthesizing polypeptides substantiallysimilar to the polypeptide. The term “substantially similar” to thepolypeptide refers to non-naturally occurring forms of the polypeptide.These polypeptides may differ in some engineered way from thepolypeptide isolated from its native source, e.g., variants that differin specific activity, thermostability, pH optimum, or the like. Thevariants may be constructed on the basis of the polynucleotide presentedas the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO:5, or the cDNA sequence thereof, by introduction of nucleotidesubstitutions that do not result in a change in the amino acid sequenceof the polypeptide, but which correspond to the codon usage of the hostorganism intended for production of the enzyme, or by introduction ofnucleotide substitutions that may give rise to a different amino acidsequence. For a general description of nucleotide substitution, see,e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of the polypeptide. Manipulation of the polynucleotideprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotides utilizing recombinant DNA methods are well known in theart.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and mutant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase Ill,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding thepolypeptide would be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA(phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB(phosphoribosylaminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris a hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAM111permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and optionally (b) recovering thepolypeptide. In one aspect, the cell is a Rasamsonia cell. In anotheraspect, the cell is a Rasamsonia byssochlamydoides cell. In anotheraspect, the cell is a Rasamsonia byssochlamydoides CBS 413.71 cell. Inanother aspect, the cell is a Rasamsonia byssochlamydoides CBS 150.75cell.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and optionally (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cells may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, a whole fermentation broth comprising apolypeptide having xylanase activity of the present invention isrecovered.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideto express and produce a polypeptide or domain of the present inventionin recoverable quantities. The polypeptide or domain may be recoveredfrom the plant or plant part. Alternatively, the plant or plant partcontaining the polypeptide or domain may be used as such for improvingthe quality of a food or feed, e.g., improving nutritional value,palatability, and rheological properties, or to destroy an antinutritivefactor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing the polypeptide or domainmay be constructed in accordance with methods known in the art. Inshort, the plant or plant cell is constructed by incorporating one ormore expression constructs encoding the polypeptide or domain into theplant host genome or chloroplast genome and propagating the resultingmodified plant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide or domain operablylinked with appropriate regulatory sequences required for expression ofthe polynucleotide in the plant or plant part of choice. Furthermore,the expression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide or domainis desired to be expressed (Sticklen, 2008, Nature Rev. 9: 433-443). Forinstance, the expression of the gene encoding a polypeptide or domainmay be constitutive or inducible, or may be developmental, stage ortissue specific, and the gene product may be targeted to a specifictissue or plant part such as seeds or leaves. Regulatory sequences are,for example, described by Tague et al., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide or domain in the plant. For instance, thepromoter enhancer element may be an intron that is placed between thepromoter and the polynucleotide encoding a polypeptide or domain. Forinstance, Xu et al., 1993, supra, disclose the use of the first intronof the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are hereinincorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a polypeptide or domain canbe introduced into a particular plant variety by crossing, without theneed for ever directly transforming a plant of that given variety.Therefore, the present invention encompasses not only a plant directlyregenerated from cells which have been transformed in accordance withthe present invention, but also the progeny of such plants. As usedherein, progeny may refer to the offspring of any generation of a parentplant prepared in accordance with the present invention. Such progenymay include a DNA construct prepared in accordance with the presentinvention. Crossing results in the introduction of a transgene into aplant line by cross pollinating a starting line with a donor plant line.Non-limiting examples of such steps are described in U.S. Pat. No.7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideor domain of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide or domain under conditions conducive for production ofthe polypeptide or domain; and (b) recovering the polypeptide or domain.

Removal or Reduction of Xylanase Activity

The present invention also relates to methods of producing a mutant of aparent cell, which comprises disrupting or deleting a polynucleotide, ora portion thereof, encoding a polypeptide of the present invention,which results in the mutant cell producing less of the polypeptide thanthe parent cell when cultivated under the same conditions.

The mutant cell may be constructed by reducing or eliminating expressionof the polynucleotide using methods well known in the art, for example,insertions, disruptions, replacements, or deletions. In a preferredaspect, the polynucleotide is inactivated. The polynucleotide to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required forexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thepolynucleotide. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the polynucleotide may be performed bysubjecting the parent cell to mutagenesis and selecting for mutant cellsin which expression of the polynucleotide has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the polynucleotide may be accomplishedby insertion, substitution, or deletion of one or more nucleotides inthe gene or a regulatory element required for transcription ortranslation thereof. For example, nucleotides may be inserted or removedso as to result in the introduction of a stop codon, the removal of thestart codon, or a change in the open reading frame. Such modification orinactivation may be accomplished by site-directed mutagenesis or PCRgenerated mutagenesis in accordance with methods known in the art.Although, in principle, the modification may be performed in vivo, i.e.,directly on the cell expressing the polynucleotide to be modified, it ispreferred that the modification be performed in vitro as exemplifiedbelow.

An example of a convenient way to eliminate or reduce expression of apolynucleotide is based on techniques of gene replacement, genedeletion, or gene disruption. For example, in the gene disruptionmethod, a nucleic acid sequence corresponding to the endogenouspolynucleotide is mutagenized in vitro to produce a defective nucleicacid sequence that is then transformed into the parent cell to produce adefective gene. By homologous recombination, the defective nucleic acidsequence replaces the endogenous polynucleotide. It may be desirablethat the defective polynucleotide also encodes a marker that may be usedfor selection of transformants in which the polynucleotide has beenmodified or destroyed. In an aspect, the polynucleotide is disruptedwith a selectable marker such as those described herein.

The present invention also relates to methods of inhibiting theexpression of a polypeptide having xylanase activity in a cell,comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. In a preferredaspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length.

The dsRNA is preferably a small interfering RNA (sRNA) or a micro RNA(miRNA). In a preferred aspect, the dsRNA is small interfering RNA forinhibiting transcription. In another preferred aspect, the dsRNA ismicro RNA for inhibiting translation.

The present invention also relates to such double-stranded RNA (dsRNA)molecules, comprising a portion of the mature polypeptide codingsequence of SEQ ID NO: 1 or of SEQ ID NO: 5 for inhibiting expression ofthe polypeptide in a cell. While the present invention is not limited byany particular mechanism of action, the dsRNA can enter a cell and causethe degradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed to dsRNA,mRNA from the homologous gene is selectively degraded by a processcalled RNA interference (RNAi).

The dsRNAs of the present invention can be used in gene-silencing. Inone aspect, the invention provides methods to selectively degrade RNAusing a dsRNAi of the present invention. The process may be practiced invitro, ex vivo or in vivo. In one aspect, the dsRNA molecules can beused to generate a loss-of-function mutation in a cell, an organ or ananimal. Methods for making and using dsRNA molecules to selectivelydegrade RNA are well known in the art; see, for example, U.S. Pat. Nos.6,489,127; 6,506,559; 6,511,824; and 6,515,109.

The present invention further relates to a mutant cell of a parent cellthat comprises a disruption or deletion of a polynucleotide encoding thepolypeptide or a control sequence thereof or a silenced gene encodingthe polypeptide, which results in the mutant cell producing less of thepolypeptide or no polypeptide compared to the parent cell.

The polypeptide-deficient mutant cells are particularly useful as hostcells for expression of native and heterologous polypeptides. Therefore,the present invention further relates to methods of producing a nativeor heterologous polypeptide, comprising (a) cultivating the mutant cellunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide. The term “heterologous polypeptides” meanspolypeptides that are not native to the host cell, e.g., a variant of anative protein. The host cell may comprise more than one copy of apolynucleotide encoding the native or heterologous polypeptide.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallyxylanase-free product are of particular interest in the production ofeukaryotic polypeptides, in particular fungal proteins such as enzymes.The xylanase-deficient cells may also be used to express heterologousproteins of pharmaceutical interest such as hormones, growth factors,receptors, and the like. The term “eukaryotic polypeptides” includes notonly native polypeptides, but also those polypeptides, e.g., enzymes,which have been modified by amino acid substitutions, deletions oradditions, or other such modifications to enhance activity,thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from xylanase activity that is produced by a method ofthe present invention.

Fermentation Broth Formulations or Cell Compositions

The present invention also relates to a fermentation broth formulationor a cell composition comprising a polypeptide of the present invention.The fermentation broth product further comprises additional ingredientsused in the fermentation process, such as, for example, cells(including, the host cells containing the gene encoding the polypeptideof the present invention which are used to produce the polypeptide ofinterest), cell debris, biomass, fermentation media and/or fermentationproducts. In some embodiments, the composition is a cell-killed wholebroth containing organic acid(s), killed cells and/or cell debris, andculture medium.

The term “fermentation broth” as used herein refers to a preparationproduced by cellular fermentation that undergoes no or minimal recoveryand/or purification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In an embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a specific embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compostions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The fermentation broth formulations or cell compositions may furthercomprise multiple enzymatic activities, such as one or more (e.g.,several) enzymes selected from the group consisting of a cellulase, ahemicellulase, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin. Thefermentation broth formulations or cell compositions may also compriseone or more (e.g., several) enzymes selected from the group consistingof a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase, or atransferase, e.g., an alpha-galactosidase, alpha-glucosidase,aminopeptidase, amylase, beta-galactosidase, beta-glucosidase,beta-xylosidase, carbohydrase, carboxypeptidase, catalase,cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, endoglucanase, esterase,glucoamylase, invertase, laccase, lipase, mannosidase, mutanase,oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis (e.g., expression of cellulase and/or glucosidase enzyme(s)).In some embodiments, the cell-killed whole broth or composition containsthe spent cell culture medium, extracellular enzymes, and killedfilamentous fungal cells. In some embodiments, the microbial cellspresent in the cell-killed whole broth or composition can bepermeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Enzyme Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that thexylanase activity of the composition has been increased, e.g., with anenrichment factor of at least 1.1.

The compositions may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the compositions may comprise multiple enzymaticactivities, such as one or more (e.g., several) enzymes selected fromthe group consisting of a cellulase, a hemicellulase, a GH61 polypeptidehaving cellulolytic enhancing activity, an esterase, an expansin, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin. The compositions may also comprise one or more (e.g.,several) enzymes selected from the group consisting of a hydrolase, anisomerase, a ligase, a lyase, an oxidoreductase, or a transferase, e.g.,an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase,beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, glucoamylase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase, or xylanase.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Thecompositions may be stabilized in accordance with methods known in theart.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Uses

The present invention is also directed to the following processes forusing the polypeptides having xylanase activity, or compositionsthereof.

The present invention also relates to processes for degrading acellulosic or xylan-containing material, comprising: treating thecellulosic or xylan-containing material with an enzyme composition inthe presence of a polypeptide having xylanase activity of the presentinvention. In one aspect, the processes further comprise recovering thedegraded cellulosic or xylan-containing material. Soluble products ofdegradation or conversion of the cellulosic or xylan-containing materialcan be separated from insoluble cellulosic or xylan-containing materialusing a method known in the art such as, for example, centrifugation,filtration, or gravity settling.

The present invention also relates to processes of producing afermentation product, comprising: (a) saccharifying a cellulosic orxylan-containing material with an enzyme composition in the presence ofa polypeptide having xylanase activity of the present invention; (b)fermenting the saccharified cellulosic or xylan-containing material withone or more (e.g., several) fermenting microorganisms to produce thefermentation product; and (c) recovering the fermentation product fromthe fermentation.

The present invention also relates to processes of fermenting acellulosic or xylan-containing material, comprising: fermenting thecellulosic or xylan-containing material with one or more (e.g., several)fermenting microorganisms, wherein the cellulosic or xylan-containingmaterial is saccharified with an enzyme composition in the presence of apolypeptide having xylanase activity of the present invention. In oneaspect, the fermenting of the cellulosic or xylan-containing materialproduces a fermentation product. In another aspect, the processesfurther comprise recovering the fermentation product from thefermentation.

The processes of the present invention can be used to saccharify thecellulosic or xylan-containing material to fermentable sugars and toconvert the fermentable sugars to many useful fermentation products,e.g., fuel (ethanol, n-butanol, isobutanol, biodiesel, jet fuel) and/orplatform chemicals (e.g., acids, alcohols, ketones, gases, oils, and thelike). The production of a desired fermentation product from thecellulosic or xylan-containing material typically involves pretreatment,enzymatic hydrolysis (saccharification), and fermentation.

The processing of the cellulosic or xylan-containing material accordingto the present invention can be accomplished using methods conventionalin the art. Moreover, the processes of the present invention can beimplemented using any conventional biomass processing apparatusconfigured to operate in accordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and co-fermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC), also sometimes calledconsolidated bioprocessing (CBP). SHF uses separate process steps tofirst enzymatically hydrolyze the cellulosic material to fermentablesugars, e.g., glucose, cellobiose, and pentose monomers, and thenferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of the cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the co-fermentation of multiple sugars (Sheehanand Himmel, 1999, Biotechnol. Prog. 15: 817-827). HHF involves aseparate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more (e.g.,several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd et al., 2002,Microbiol. Mol. Biol. Reviews 66: 506-577). It is understood herein thatany method known in the art comprising pretreatment, enzymatichydrolysis (saccharification), fermentation, or a combination thereof,can be used in the practicing the processes of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (deCastilhos Corazza et al., 2003, Acta Scientiarum. Technology 25: 33-38;Gusakov and Sinitsyn, 1985, Enz. Microb. Technol. 7: 346-352), anattrition reactor (Ryu and Lee, 1983, Biotechnol. Bioeng. 25: 53-65).Additional reactor types include fluidized bed, upflow blanket,immobilized, and extruder type reactors for hydrolysis and/orfermentation.

Pretreatment.

In practicing the processes of the present invention, any pretreatmentprocess known in the art can be used to disrupt plant cell wallcomponents of the cellulosic or xylan-containing material (Chandra etal., 2007, Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe andZacchi, 2007, Adv. Biochem. Engin./Biotechnol. 108: 41-65; Hendriks andZeeman, 2009, Bioresource Technology 100: 10-18; Mosier et al., 2005,Bioresource Technology 96: 673-686; Taherzadeh and Karimi, 2008, Int. J.Mol. Sci. 9: 1621-1651; Yang and Wyman, 2008, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic or xylan-containing material can also be subjected toparticle size reduction, sieving, pre-soaking, wetting, washing, and/orconditioning prior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, ionic liquid, and gammairradiation pretreatments.

The cellulosic or xylan-containing material can be pretreated beforehydrolysis and/or fermentation. Pretreatment is preferably performedprior to the hydrolysis. Alternatively, the pretreatment can be carriedout simultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, the cellulosic orxylan-containing material is heated to disrupt the plant cell wallcomponents, including lignin, hemicellulose, and cellulose to make thecellulose and other fractions, e.g., hemicellulose, accessible toenzymes. The cellulosic or xylan-containing material is passed to orthrough a reaction vessel where steam is injected to increase thetemperature to the required temperature and pressure and is retainedtherein for the desired reaction time. Steam pretreatment is preferablyperformed at 140-250° C., e.g., 160-200° C. or 170-190° C., where theoptimal temperature range depends on optional addition of a chemicalcatalyst. Residence time for the steam pretreatment is preferably 1-60minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12 minutes, or 4-10minutes, where the optimal residence time depends on the temperature andoptional addition of a chemical catalyst. Steam pretreatment allows forrelatively high solids loadings, so that the cellulosic orxylan-containing material is generally only moist during thepretreatment. The steam pretreatment is often combined with an explosivedischarge of the material after the pretreatment, which is known assteam explosion, that is, rapid flashing to atmospheric pressure andturbulent flow of the material to increase the accessible surface areaby fragmentation (Duff and Murray, 1996, Bioresource Technology 855:1-33; Galbe and Zacchi, 2002, Appl. Microbiol. Biotechnol. 59: 618-628;U.S. Patent Application No. 2002/0164730). During steam pretreatment,hemicellulose acetyl groups are cleaved and the resulting acidautocatalyzes partial hydrolysis of the hemicellulose to monosaccharidesand oligosaccharides. Lignin is removed to only a limited extent.

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Such a pretreatment can convertcrystalline cellulose to amorphous cellulose. Examples of suitablechemical pretreatment processes include, for example, dilute acidpretreatment, lime pretreatment, wet oxidation, ammonia fiber/freezeexpansion (AFEX), ammonia percolation (APR), ionic liquid, andorganosolv pretreatments.

A chemical catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 5% w/w) issometimes added prior to steam pretreatment, which decreases the timeand temperature, increases the recovery, and improves enzymatichydrolysis (Ballesteros et al., 2006, Appl. Biochem. Biotechnol.129-132: 496-508; Varga et al., 2004, Appl. Biochem. Biotechnol.113-116: 509-523; Sassner et al., 2006, Enzyme Microb. Technol. 39:756-762). In dilute acid pretreatment, the cellulosic material is mixedwith dilute acid, typically H₂SO₄, and water to form a slurry, heated bysteam to the desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, supra; Schell et al., 2004, Bioresource Technology 91:179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to,sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), andammonia fiber/freeze expansion (AFEX) pretreatment.

Lime pretreatment is performed with calcium oxide or calcium hydroxideat temperatures of 85-150° C. and residence times from 1 hour to severaldays (Wyman et al., 2005, Bioresource Technology 96: 1959-1966; Mosieret al., 2005, Bioresource Technology 96: 673-686). WO 2006/110891, WO2006/110899, WO 2006/110900, and WO 2006/110901 disclose pretreatmentmethods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technology 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed preferably at 1-40% drymatter, e.g., 2-30% dry matter or 5-20% dry matter, and often theinitial pH is increased by the addition of alkali such as sodiumcarbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion) can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber expansion (AFEX) involves treating the cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-150°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technology 96:2014-2018). During AFEX pretreatment cellulose and hemicelluloses remainrelatively intact. Lignin-carbohydrate complexes are cleaved.

Organosolv pretreatment delignifies the cellulosic material byextraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose andlignin is removed.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. Biotechnol. 105-108: 69-85, and Mosier etal., 2005, Bioresource Technology 96: 673-686, and U.S. application no.2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as adilute acid treatment, and more preferably as a continuous dilute acidtreatment. The acid is typically sulfuric acid, but other acids can alsobe used, such as acetic acid, citric acid, nitric acid, phosphoric acid,tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof.Mild acid treatment is conducted in the pH range of preferably 1-5,e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in therange from preferably 0.01 to 10 wt. % acid, e.g., 0.05 to 5 wt. % acidor 0.1 to 2 wt. % acid. The acid is contacted with the cellulosic orxylan-containing material and held at a temperature in the range ofpreferably 140-200° C., e.g., 165-190° C., for periods ranging from 1 to60 minutes.

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic or xylan-containing material ispresent during pretreatment in amounts preferably between 10-80 wt. %,e.g., 20-70 wt. % or 30-60 wt. %, such as around 40 wt. %. Thepretreated cellulosic or xylan-containing material can be unwashed orwashed using any method known in the art, e.g., washed with water.

Mechanical Pretreatment or Physical Pretreatment: The term “mechanicalpretreatment” or “physical pretreatment” refers to any pretreatment thatpromotes size reduction of particles. For example, such pretreatment caninvolve various types of grinding or milling (e.g., dry milling, wetmilling, or vibratory ball milling).

The cellulosic or xylan-containing material can be pretreated bothphysically (mechanically) and chemically. Mechanical or physicalpretreatment can be coupled with steaming/steam explosion,hydrothermolysis, dilute or mild acid treatment, high temperature, highpressure treatment, irradiation (e.g., microwave irradiation), orcombinations thereof. In one aspect, high pressure means pressure in therange of preferably about 100 to about 400 psi, e.g., about 150 to about250 psi. In another aspect, high temperature means temperature in therange of about 100 to about 300° C., e.g., about 140 to about 200° C. Ina preferred aspect, mechanical or physical pretreatment is performed ina batch-process using a steam gun hydrolyzer system that uses highpressure and high temperature as defined above, e.g., a Sunds Hydrolyzeravailable from Sunds Defibrator AB, Sweden. The physical and chemicalpretreatments can be carried out sequentially or simultaneously, asdesired.

Accordingly, in a preferred aspect, the cellulosic or xylan-containingmaterial is subjected to physical (mechanical) or chemical pretreatment,or any combination thereof, to promote the separation and/or release ofcellulose, hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic orxylan-containing material. Biological pretreatment techniques caninvolve applying lignin-solubilizing microorganisms and/or enzymes (see,for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook onBioethanol: Production and Utilization, Wyman, C. E., ed., Taylor &Francis, Washington, D.C., 179-212; Ghosh and Singh, 1993, Adv. Appl.Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreatinglignocellulosic biomass: a review, in Enzymatic Conversion of Biomassfor Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P.,eds., ACS Symposium Series 566, American Chemical Society, Washington,D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T.,1999, Ethanol production from renewable resources, in Advances inBiochemical Engineering/Biotechnology, Scheper, T., ed., Springer-VerlagBerlin Heidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996,Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990, Adv.Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification.

In the hydrolysis step, also known as saccharification, the cellulosicor xylan-containing material, e.g., pretreated, is hydrolyzed to breakdown cellulose and/or hemicellulose to fermentable sugars, such asglucose, cellobiose, xylose, xylulose, arabinose, mannose, galactose,and/or soluble oligosaccharides. The hydrolysis is performedenzymatically by an enzyme composition in the presence of a polypeptidehaving xylanase activity of the present invention. The enzymes of thecompositions can be added simultaneously or sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In one aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the cellulosic or xylan-containing material isfed gradually to, for example, an enzyme containing hydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 120 hours, e.g., about 16 to about 72 hours or about24 to about 48 hours. The temperature is in the range of preferablyabout 25° C. to about 70° C., e.g., about 30° C. to about 65° C., about40° C. to about 60° C., or about 50° C. to about 55° C. The pH is in therange of preferably about 3 to about 8, e.g., about 3.5 to about 7,about 4 to about 6, or about 4.0 to about 5.5. The dry solids content isin the range of preferably about 5 to about 50 wt. %, e.g., about 10 toabout 40 wt. % or about 20 to about 30 wt. %.

The enzyme compositions can comprise any protein useful in degrading thecellulosic or xylan-containing material.

In one aspect, the enzyme composition comprises or further comprises oneor more (e.g., several) proteins selected from the group consisting of acellulase, a GH61 polypeptide, a hemicellulase, an esterase, anexpansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, aprotease, and a swollenin. In another aspect, the cellulase ispreferably one or more (e.g., several) enzymes selected from the groupconsisting of an endoglucanase, a cellobiohydrolase, and abeta-glucosidase. In another aspect, the hemicellulase is preferably oneor more (e.g., several) enzymes selected from the group consisting of anacetylmannan esterase, an acetylxylan esterase, an arabinanase, anarabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, agalactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, amannosidase, a xylanase, and a xylosidase.

In another aspect, the enzyme composition comprises one or more (e.g.,several) cellulolytic enzymes. In another aspect, the enzyme compositioncomprises or further comprises one or more (e.g., several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (e.g., several) cellulolytic enzymes and one ormore (e.g., several) hemicellulolytic enzymes. In another aspect, theenzyme composition comprises one or more (e.g., several) enzymesselected from the group of cellulolytic enzymes and hemicellulolyticenzymes. In another aspect, the enzyme composition comprises anendoglucanase. In another aspect, the enzyme composition comprises acellobiohydrolase. In another aspect, the enzyme composition comprises abeta-glucosidase. In another aspect, the enzyme composition comprises aGH61 polypeptide. In another aspect, the enzyme composition comprises anendoglucanase and a GH61 polypeptide. In another aspect, the enzymecomposition comprises a cellobiohydrolase and a GH61 polypeptide. Inanother aspect, the enzyme composition comprises a beta-glucosidase anda GH61 polypeptide. In another aspect, the enzyme composition comprisesan endoglucanase and a cellobiohydrolase. In another aspect, the enzymecomposition comprises an endoglucanase and a cellobiohydrolase I, acellobiohydrolase II, or a combination of a cellobiohydrolase I and acellobiohydrolase II. In another aspect, the enzyme compositioncomprises an endoglucanase and a beta-glucosidase. In another aspect,the enzyme composition comprises a beta-glucosidase and acellobiohydrolase. In another aspect, the enzyme composition comprises abeta-glucosidase and a cellobiohydrolase I, a cellobiohydrolase II, or acombination of a cellobiohydrolase I and a cellobiohydrolase II Inanother aspect, the enzyme composition comprises an endoglucanase, aGH61 polypeptide, and a cellobiohydrolase. In another aspect, the enzymecomposition comprises an endoglucanase, a GH61 polypeptide, and acellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II. In another aspect, theenzyme composition comprises an endoglucanase, a beta-glucosidase, and aGH61 polypeptide. In another aspect, the enzyme composition comprises abeta-glucosidase, a GH61 polypeptide, and a cellobiohydrolase. Inanother aspect, the enzyme composition comprises a beta-glucosidase, aGH61 polypeptide, and a cellobiohydrolase I, a cellobiohydrolase II, ora combination of a cellobiohydrolase I and a cellobiohydrolase II. Inanother aspect, the enzyme composition comprises an endoglucanase, abeta-glucosidase, and a cellobiohydrolase. In another aspect, the enzymecomposition comprises an endoglucanase, a beta-glucosidase, and acellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II. In another aspect, theenzyme composition comprises an endoglucanase, a cellobiohydrolase, abeta-glucosidase, and a GH61 polypeptide. In another aspect, the enzymecomposition comprises an endoglucanase, a beta-glucosidase, a GH61polypeptide, and a cellobiohydrolase I, a cellobiohydrolase II, or acombination of a cellobiohydrolase I and a cellobiohydrolase II.

In another aspect, the enzyme composition comprises an acetylmannanesterase. In another aspect, the enzyme composition comprises anacetylxylan esterase. In another aspect, the enzyme compositioncomprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect,the enzyme composition comprises an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase). In another aspect, the enzyme compositioncomprises a coumaric acid esterase. In another aspect, the enzymecomposition comprises a feruloyl esterase. In another aspect, the enzymecomposition comprises a galactosidase (e.g., alpha-galactosidase and/orbeta-galactosidase). In another aspect, the enzyme composition comprisesa glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, theenzyme composition comprises a glucuronoyl esterase. In another aspect,the enzyme composition comprises a mannanase. In another aspect, theenzyme composition comprises a mannosidase (e.g., beta-mannosidase). Inanother aspect, the enzyme composition comprises a xylanase. In apreferred aspect, the xylanase is a Family 10 xylanase. In anotheraspect, the enzyme composition comprises a xylosidase (e.g.,beta-xylosidase).

In another aspect, the enzyme composition comprises an esterase. Inanother aspect, the enzyme composition comprises an expansin. In anotheraspect, the enzyme composition comprises a ligninolytic enzyme. In apreferred aspect, the ligninolytic enzyme is a manganese peroxidase. Inanother preferred aspect, the ligninolytic enzyme is a ligninperoxidase. In another preferred aspect, the ligninolytic enzyme is aH₂O₂-producing enzyme. In another aspect, the enzyme compositioncomprises a pectinase. In another aspect, the enzyme compositioncomprises an oxidoreductase. In another aspect, the enzyme compositioncomprises a protease. In another aspect, the enzyme compositioncomprises a swollenin.

In the processes of the present invention, the enzyme(s) can be addedprior to or during saccharification, saccharification and fermentation,or fermentation.

One or more (e.g., several) components of the enzyme composition may benative proteins, recombinant proteins, or a combination of nativeproteins and recombinant proteins. For example, one or more (e.g.,several) components may be native proteins of a cell, which is used as ahost cell to express recombinantly one or more (e.g., several) othercomponents of the enzyme composition. It is understood herein that therecombinant proteins may be heterologous (e.g., foreign) and native tothe host cell. One or more (e.g., several) components of the enzymecomposition may be produced as monocomponents, which are then combinedto form the enzyme composition. The enzyme composition may be acombination of multicomponent and monocomponent protein preparations.

The enzymes used in the processes of the present invention may be in anyform suitable for use, such as, for example, a fermentation brothformulation or a cell composition, a cell lysate with or withoutcellular debris, a semi-purified or purified enzyme preparation, or ahost cell as a source of the enzymes. The enzyme composition may be adry powder or granulate, a non-dusting granulate, a liquid, a stabilizedliquid, or a stabilized protected enzyme. Liquid enzyme preparationsmay, for instance, be stabilized by adding stabilizers such as a sugar,a sugar alcohol or another polyol, and/or lactic acid or another organicacid according to established processes.

The optimum amounts of the enzymes and polypeptides having xylanaseactivity depend on several factors including, but not limited to, themixture of cellulolytic enzymes and/or hemicellulolytic enzymes, thecellulosic or xylan-containing material, the concentration of cellulosicor xylan-containing material, the pretreatment(s) of the cellulosic orxylan-containing material, temperature, time, pH, and inclusion of afermenting organism (e.g., for Simultaneous Saccharification andFermentation.

In one aspect, an effective amount of cellulolytic or hemicellulolyticenzyme to the cellulosic or xylan-containing material is about 0.5 toabout 50 mg, e.g., about 0.5 to about 40 mg, about 0.5 to about 25 mg,about 0.75 to about 20 mg, about 0.75 to about 15 mg, about 0.5 to about10 mg, or about 2.5 to about 10 mg per g of the cellulosic orxylan-containing material.

In another aspect, an effective amount of a polypeptide having xylanaseactivity to the cellulosic or xylan-containing material is about 0.01 toabout 50.0 mg, e.g., about 0.01 to about 40 mg, about 0.01 to about 30mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01 toabout 5 mg, about 0.025 to about 1.5 mg, about 0.05 to about 1.25 mg,about 0.075 to about 1.25 mg, about 0.1 to about 1.25 mg, about 0.15 toabout 1.25 mg, or about 0.25 to about 1.0 mg per g of the cellulosic orxylan-containing material.

In another aspect, an effective amount of a polypeptide having xylanaseactivity to cellulolytic or hemicellulolytic enzyme is about 0.005 toabout 1.0 g, e.g., about 0.01 to about 1.0 g, about 0.15 to about 0.75g, about 0.15 to about 0.5 g, about 0.1 to about 0.5 g, about 0.1 toabout 0.25 g, or about 0.05 to about 0.2 g per g of cellulolytic orhemicellulolytic enzyme.

The polypeptides having cellulolytic enzyme activity or hemicellulolyticenzyme activity as well as other proteins/polypeptides useful in thedegradation of the cellulosic or xylan-containing material (collectivelyhereinafter “polypeptides having enzyme activity”) can be derived orobtained from any suitable origin, including, archaeal, bacterial,fungal, yeast, plant, or animal origin. The term “obtained” also meansherein that the enzyme may have been produced recombinantly in a hostorganism employing methods described herein, wherein the recombinantlyproduced enzyme is either native or foreign to the host organism or hasa modified amino acid sequence, e.g., having one or more (e.g., several)amino acids that are deleted, inserted and/or substituted, i.e., arecombinantly produced enzyme that is a mutant and/or a fragment of anative amino acid sequence or an enzyme produced by nucleic acidshuffling processes known in the art. Encompassed within the meaning ofa native enzyme are natural variants and within the meaning of a foreignenzyme are variants obtained by, e.g., site-directed mutagenesis orshuffling.

A polypeptide having enzyme activity may be a bacterial polypeptide. Forexample, the polypeptide may be a Gram-positive bacterial polypeptidehaving enzyme activity, or a Gram-negative bacterial polypeptide havingenzyme activity.

The polypeptide having enzyme activity may also be a fungal polypeptide,and more preferably a yeast polypeptide having enzyme activity or morepreferably a filamentous fungal polypeptide having enzyme activity.

Chemically modified or protein engineered mutants of polypeptides havingenzyme activity may also be used.

One or more (e.g., several) components of the enzyme composition may bea recombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host is preferably a heterologous host (enzyme isforeign to host), but the host may under certain conditions also be ahomologous host (enzyme is native to host). Monocomponent cellulolyticproteins may also be prepared by purifying such a protein from afermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymescomprise a commercial cellulolytic enzyme preparation. Examples ofcommercial cellulolytic enzyme preparations suitable for use in thepresent invention include, for example, CELLIC® CTec (Novozymes NS),CELLIC® CTec2 (Novozymes NS), CELLIC® CTec3 (Novozymes NS), CELLUCLAST™(Novozymes NS), NOVOZYM™ 188 (Novozymes NS), SPEZYME™ CP (GenencorInt.), ACCELERASE™ TRIO (DuPont), FILTRASE® NL (DSM); METHAPLUS® S/L 100(DSM), ROHAMENT™ 7069 W (Rohm GmbH), or ALTERNAFUEL® CMAX3™ (DyadicInternational, Inc.). The cellulase enzymes are added in amountseffective from about 0.001 to about 5.0 wt. % of solids, e.g., about0.025 to about 4.0 wt. % of solids or about 0.005 to about 2.0 wt. % ofsolids.

Examples of bacterial endoglucanases that can be used in the processesof the present invention, include, but are not limited to, Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655; WO 00/70031; WO05/093050), Erwinia carotovara endoglucanase (Saarilahti et al., 1990,Gene 90: 9-14), Thermobifida fusca endoglucanase III (WO 05/093050), andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention, include, but are not limited to, Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichodermareesei Cel7B endoglucanase I (GenBank:M15665), Trichoderma reeseiendoglucanase II (Saloheimo et al., 1988, Gene 63:11-22), Trichodermareesei Cel5A endoglucanase II (GenBank:M19373), Trichoderma reeseiendoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol. 64:555-563, GenBank:AB003694), Trichoderma reesei endoglucanase V(Saloheimo et al., 1994, Molecular Microbiology 13: 219-228,GenBank:Z33381), Aspergillus aculeatus endoglucanase (Ooi et al., 1990,Nucleic Acids Research 18: 5884), Aspergillus kawachii endoglucanase(Sakamoto et al., 1995, Current Genetics 27: 435-439), Fusariumoxysporum endoglucanase (GenBank:L29381), Humicola grisea var.thermoidea endoglucanase (GenBank:AB003107), Melanocarpus albomycesendoglucanase (GenBank:MAL515703), Neurospora crassa endoglucanase (GenBank:XM_324477), Humicola insolens endoglucanase V, Myceliophthorathermophila CBS 117.65 endoglucanase, Thermoascus aurantiacusendoglucanase I (GenBank:AF487830) and Trichoderma reesei strain No.VTT-D-80133 endoglucanase (GenBank:M15665).

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Aspergillus aculeatus cellobiohydrolase II (WO2011/059740), Chaetomium thermophilum cellobiohydrolase I, Chaetomiumthermophilum cellobiohydrolase II, Humicola insolens cellobiohydrolaseI, Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871),Penicillium occitanis cellobiohydrolase I (Gen Bank:AY690482),Talaromyces emersonii cellobiohydrolase I (GenBank:AF439936), Thielaviahyrcanie cellobiohydrolase II (WO 2010/141325), Thielavia terrestriscellobiohydrolase II (CEL6A, WO 2006/074435), Trichoderma reeseicellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, andTrichophaea saccata cellobiohydrolase II (WO 2010/057086).

Examples of beta-glucosidases useful in the present invention include,but are not limited to, beta-glucosidases from Aspergillus aculeatus(Kawaguchi et al., 1996, Gene 173: 287-288), Aspergillus fumigatus (WO2005/047499), Aspergillus niger (Dan et al., 2000, J. Biol. Chem. 275:4973-4980), Aspergillus oryzae (WO 02/095014), Penicillium brasilianumIBT 20888 (WO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO2011/035029), and Trichophaea saccata (WO 2007/019442).

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat, 1991, Biochem. J. 280: 309-316,and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.

In the processes of the present invention, any GH61 polypeptide havingcellulolytic enhancing activity can be used as a component of the enzymecomposition.

Examples of GH61 polypeptides useful in the processes of the presentinvention include, but are not limited to, GH61 polypeptides fromThielavia terrestris (WO 2005/074647, WO 2008/148131, and WO2011/035027), Thermoascus aurantiacus (WO 2005/074656 and WO2010/065830), Trichoderma reesei (WO 2007/089290), Myceliophthorathermophila (WO 2009/085935, WO 2009/085859, WO 2009/085864, and WO2009/085868), Aspergillus fumigatus (WO 2010/138754), Penicilliumpinophilum (WO 2011/005867), Thermoascus sp. (WO 2011/039319),Penicillium sp. (WO 2011/041397), Thermoascus crustaceous (WO2011/041504), Aspergillus aculeatus (WO 2012/125925), Thermomyceslanuginosus (WO 2012/113340, WO 12/129699, and WO 2012/130964),Aurantiporus alborubescens (WO 2012/122477), Trichophaea saccata (WO2012/122477), Penicillium thomii (WO 2012/122477), Talaromycesstipitatus (WO 2012/135659), Humicola insolens (WO 2012/146171),Malbranchea cinnamomea (WO 2012/101206), Talaromyces leycettanus (WO2012/101206), and Chaetomium thermophilum (WO 2012/101206).

In one aspect, the GH61 polypeptide is used in the presence of a solubleactivating divalent metal cation according to WO 2008/151043, e.g.,manganese or copper.

In another aspect, the GH61 polypeptide is used in the presence of adioxy compound, a bicylic compound, a heterocyclic compound, anitrogen-containing compound, a quinone compound, a sulfur-containingcompound, or a liquor obtained from a pretreated cellulosic materialsuch as pretreated corn stover (WO 2012/021394, WO 2012/021395, WO2012/021396, WO 2012/021399, WO 2012/021400, WO 2012/021401, WO2012/021408, and WO 2012/021410).

In one aspect, such a compound is added at a molar ratio of the compoundto glucosyl units of cellulose of about 10⁻⁶ to about 10, e.g., about10⁻⁶ to about 7.5, about 10⁻⁶ to about 5, about 10⁻⁶ to about 2.5, about10⁻⁶ to about 1, about 10⁻⁶ to about 1, about 10⁻⁶ to about 10⁻¹, about10⁻⁴ to about 10⁻¹, about 10⁻³ to about 10⁻¹, or about 10⁻³ to about10⁻². In another aspect, an effective amount of such a compound is about0.1 μM to about 1 M, e.g., about 0.5 μM to about 0.75 M, about 0.75 μMto about 0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M,about 5 μM to about 50 mM, about 10 μM to about 25 mM, about 50 μM toabout 25 mM, about 10 μM to about 10 mM, about 5 μM to about 5 mM, orabout 0.1 mM to about 1 mM.

The term “liquor” means the solution phase, either aqueous, organic, ora combination thereof, arising from treatment of a lignocellulose and/orhemicellulose material in a slurry, or monosaccharides thereof, e.g.,xylose, arabinose, mannose, etc., under conditions as described herein,and the soluble contents thereof. A liquor for cellulolytic enhancementof a GH61 polypeptide can be produced by treating a lignocellulose orhemicellulose material (or feedstock) by applying heat and/or pressure,optionally in the presence of a catalyst, e.g., acid, optionally in thepresence of an organic solvent, and optionally in combination withphysical disruption of the material, and then separating the solutionfrom the residual solids. Such conditions determine the degree ofcellulolytic enhancement obtainable through the combination of liquorand a GH61 polypeptide during hydrolysis of a cellulosic substrate by acellulase preparation. The liquor can be separated from the treatedmaterial using a method standard in the art, such as filtration,sedimentation, or centrifugation.

In one aspect, an effective amount of the liquor to cellulose is about10⁻⁶ to about 10 g per g of cellulose, e.g., about 10⁻⁶ to about 7.5 g,about 10⁻⁶ to about 5 g, about 10⁻⁶ to about 2.5 g, about 10⁻⁶ to about1 g, about 10⁻⁵ to about 1 g, about 10⁻⁵ to about 10⁻¹ g, about 10⁻⁴ toabout 10⁻¹ g, about 10⁻³ to about 10⁻¹ g, or about 10⁻³ to about 10⁻² gper g of cellulose.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes NS),CELLIC® HTec (Novozymes NS), CELLIC® HTec2 (Novozymes NS), CELLIC® HTec3(Novozymes NS), VISCOZYME® (Novozymes NS), ULTRAFLO® (Novozymes NS),PULPZYME® HC (Novozymes NS), MULTIFECT® Xylanase (Genencor),ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX-200A(AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit,Wales, UK), DEPOL™ 740 L. (Biocatalysts Limit, Wales, UK), and DEPOL™762P (Biocatalysts Limit, Wales, UK), ALTERNA FUEL 100P (Dyadic), andALTERNA FUEL 200P (Dyadic).

Examples of xylanases useful in the processes of the present inventioninclude, but are not limited to, xylanases from Aspergillus aculeatus(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (WO2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium sp.(WO 2010/126772), Talaromyces lanuginosus GH11 (WO 2012/130965),Talaromyces thermophilus GH11 (WO 2012/13095), Thielavia terrestris NRRL8126 (WO 2009/079210), and Trichophaea saccata GH10 (WO 2011/057083).

Examples of beta-xylosidases useful in the processes of the presentinvention include, but are not limited to, beta-xylosidases fromNeurospora crassa (SwissProt:Q7SOW4), Trichoderma reesei(UniProtKB/TrEMBL:Q92458), Talaromyces emersonii (SwissProt:Q8X212), andTalaromyces thermophilus GH11 (WO 2012/130965).

Examples of acetylxylan esterases useful in the processes of the presentinvention include, but are not limited to, acetylxylan esterases fromAspergillus aculeatus (WO 2010/108918), Chaetomium globosum(UniProt:Q2GWX4), Chaetomium gracile (GeneSeqP:AAB82124), Humicolainsolens DSM 1800 (WO 2009/073709), Hypocrea jecorina (WO 2005/001036),Myceliophtera thermophila (WO 2010/014880), Neurospora crassa(UniProt:q7s259), Phaeosphaeria nodorum (UniProt:QOUHJ1), and Thielaviaterrestris NRRL 8126 (WO 2009/042846).

Examples of feruloyl esterases (ferulic acid esterases) useful in theprocesses of the present invention include, but are not limited to,feruloyl esterases form Humicola insolens DSM 1800 (WO 2009/076122),Neosartorya fischeri (UniProt:A1D9T4), Neurospora crassa(UniProt:Q9HGR3), Penicillium aurantiogriseum (WO 2009/127729), andThielavia terrestris (WO 2010/053838 and WO 2010/065448).

Examples of arabinofuranosidases useful in the processes of the presentinvention include, but are not limited to, arabinofuranosidases fromAspergillus niger (GeneSeqP:AAR94170), Humicola insolens DSM 1800 (WO2006/114094 and WO 2009/073383), and M. giganteus (WO 2006/114094).

Examples of alpha-glucuronidases useful in the processes of the presentinvention include, but are not limited to, alpha-glucuronidases fromAspergillus clavatus (UniProt:alcc12), Aspergillus fumigatus(SwissProt:Q4WW45), Aspergillus niger (UniProt:Q96WX9), Aspergillusterreus (SwissProt:Q0CJ P9), Humicola insolens (WO 2010/014706),Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii(UniProt:Q8X211), and Trichoderma reesei (UniProt:Q99024).

The polypeptides having enzyme activity used in the processes of thepresent invention may be produced by fermentation of the above-notedmicrobial strains on a nutrient medium containing suitable carbon andnitrogen sources and inorganic salts, using procedures known in the art(see, e.g., Bennett, J. W. and LaSure, L. (eds.), More GeneManipulations in Fungi, Academic Press, CA, 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand enzyme production are known in the art (see, e.g., Bailey, J. E.,and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill BookCompany, NY, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme or protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe enzyme to be expressed or isolated. The resulting enzymes producedby the methods described above may be recovered from the fermentationmedium and purified by conventional procedures.

Fermentation.

The fermentable sugars obtained from the hydrolyzed cellulosic orxylan-containing material can be fermented by one or more (e.g.,several) fermenting microorganisms capable of fermenting the sugarsdirectly or indirectly into a desired fermentation product.“Fermentation” or “fermentation process” refers to any fermentationprocess or any process comprising a fermentation step. Fermentationprocesses also include fermentation processes used in the consumablealcohol industry (e.g., beer and wine), dairy industry (e.g., fermenteddairy products), leather industry, and tobacco industry. Thefermentation conditions depend on the desired fermentation product andfermenting organism and can easily be determined by one skilled in theart.

In the fermentation step, sugars, released from the cellulosic orxylan-containing material as a result of the pretreatment and enzymatichydrolysis steps, are fermented to a product, e.g., ethanol, by afermenting organism, such as yeast. Hydrolysis (saccharification) andfermentation can be separate or simultaneous.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on economics, i.e., costs per equivalent sugarpotential, and recalcitrance to enzymatic conversion.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be hexose and/or pentose fermenting organisms, or acombination thereof. Both hexose and pentose fermenting organisms arewell known in the art. Suitable fermenting microorganisms are able toferment, i.e., convert, sugars, such as glucose, xylose, xylulose,arabinose, maltose, mannose, galactose, and/or oligosaccharides,directly or indirectly into the desired fermentation product. Examplesof bacterial and fungal fermenting organisms producing ethanol aredescribed by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

Examples of fermenting microorganisms that can ferment hexose sugarsinclude bacterial and fungal organisms, such as yeast. Yeast includestrains of Candida, Kluyveromyces, and Saccharomyces, e.g., Candidasonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment pentose sugars intheir native state include bacterial and fungal organisms, such as someyeast. Xylose fermenting yeast include strains of Candida, preferably C.sheatae or C. sonorensis; and strains of Pichia, e.g., P. stipitis, suchas P. stipitis CBS 5773. Pentose fermenting yeast include strains ofPachysolen, preferably P. tannophilus. Organisms not capable offermenting pentose sugars, such as xylose and arabinose, may begenetically modified to do so by methods known in the art.

Examples of bacteria that can efficiently ferment hexose and pentose toethanol include, for example, Bacillus coagulans, Clostridiumacetobutylicum, Clostridium thermocellum, Clostridium phytofermentans,Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonasmobilis (Philippidis, 1996, supra).

Other fermenting organisms include strains of Bacillus, such as Bacilluscoagulans; Candida, such as C. sonorensis, C. methanosorbosa, C.diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C.entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis,and C. scehatae; Clostridium, such as C. acetobutylicum, C.thermocellum, and C. phytofermentans; E. coli, especially E. colistrains that have been genetically modified to improve the yield ofethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K.lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such asS. pombe; Thermoanaerobacter, such as Thermoanaerobactersaccharolyticum; and Zymomonas, such as Zymomonas mobilis.

Commercially available yeast suitable for ethanol production include,e.g., BIOFERM™ AFT and XR (NABC—North American Bioproducts Corporation,GA, USA), ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™(Fleischmann's Yeast, USA), FERMIOL™ (DSM Specialties), GERT STRAND™(Gert Strand AB, Sweden), and SUPERSTART™ and THERMOSACC™ fresh yeast(Ethanol Technology, WI, USA).

In an aspect, the fermenting microorganism has been genetically modifiedto provide the ability to ferment pentose sugars, such as xyloseutilizing, arabinose utilizing, and xylose and arabinose co-utilizingmicroorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Appl.Biochem. Biotechnol. 39-40: 135-147; Ho et al., 1998, Appl. Environ.Microbiol. 64: 1852-1859; Kotter and Ciriacy, 1993, Appl. Microbiol.Biotechnol. 38: 776-783; Walfridsson et al., 1995, Appl. Environ.Microbiol. 61: 4184-4190; Kuyper et al., 2004, FEMS Yeast Research 4:655-664; Beall et al., 1991, Biotech. Bioeng. 38: 296-303; Ingram etal., 1998, Biotechnol. Bioeng. 58: 204-214; Zhang et al., 1995, Science267: 240-243; Deanda et al., 1996, Appl. Environ. Microbiol. 62:4465-4470; WO 03/062430).

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedcellulosic material or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, e.g., about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., e.g.,about 32° C. or 50° C., and about pH 3 to about pH 8, e.g., pH 4-5, 6,or 7.

In one aspect, the yeast and/or another microorganism are applied to thedegraded cellulosic material and the fermentation is performed for about12 to about 96 hours, such as typically 24-60 hours. In another aspect,the temperature is preferably between about 20° C. to about 60° C.,e.g., about 25° C. to about 50° C., about 32° C. to about 50° C., orabout 32° C. to about 50° C., and the pH is generally from about pH 3 toabout pH 7, e.g., about pH 4 to about pH 7. However, some fermentingorganisms, e.g., bacteria, have higher fermentation temperature optima.Yeast or another microorganism is preferably applied in amounts ofapproximately 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰,especially approximately 2×10⁸ viable cell count per ml of fermentationbroth. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products:

A fermentation product can be any substance derived from thefermentation. The fermentation product can be, without limitation, analcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol,methanol, ethylene glycol, 1,3-propanediol [propylene glycol],butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane,hexane, heptane, octane, nonane, decane, undecane, and dodecane), acycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, andcyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene); anamino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine,and threonine); a gas (e.g., methane, hydrogen (H₂), carbon dioxide(CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); anorganic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbicacid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaricacid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid,3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonicacid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, andxylonic acid); and polyketide. The fermentation product can also beprotein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. The alcohol can be, but is notlimited to, n-butanol, isobutanol, ethanol, methanol, arabinitol,butanediol, ethylene glycol, glycerin, glycerol, 1,3-propanediol,sorbitol, xylitol. See, for example, Gong et al., 1999, Ethanolproduction from renewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Silveira and Jonas, 2002, Appl.Microbiol. Biotechnol. 59: 400-408; Nigam and Singh, 1995, ProcessBiochemistry 30(2): 117-124; Ezeji et al., 2003, World Journal ofMicrobiology and Biotechnology 19(6): 595-603.

In another preferred aspect, the fermentation product is an alkane. Thealkane may be an unbranched or a branched alkane. The alkane can be, butis not limited to, pentane, hexane, heptane, octane, nonane, decane,undecane, or dodecane.

In another preferred aspect, the fermentation product is a cycloalkane.The cycloalkane can be, but is not limited to, cyclopentane,cyclohexane, cycloheptane, or cyclooctane.

In another preferred aspect, the fermentation product is an alkene. Thealkene may be an unbranched or a branched alkene. The alkene can be, butis not limited to, pentene, hexene, heptene, or octene.

In another preferred aspect, the fermentation product is an amino acid.The organic acid can be, but is not limited to, aspartic acid, glutamicacid, glycine, lysine, serine, or threonine. See, for example, Richardand Margaritis, 2004, Biotechnology and Bioengineering 87(4): 501-515.

In another preferred aspect, the fermentation product is a gas. The gascan be, but is not limited to, methane, H₂, CO₂, or CO. See, forexample, Kataoka et al., 1997, Water Science and Technology 36(6-7):41-47; and Gunaseelan, 1997, Biomass and Bioenergy 13(1-2): 83-114.

In another preferred aspect, the fermentation product is isoprene.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. The ketone can be, but is notlimited to, acetone.

In another preferred aspect, the fermentation product is an organicacid. The organic acid can be, but is not limited to, acetic acid,acetonic acid, adipic acid, ascorbic acid, citric acid,2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid,gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid,itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid,propionic acid, succinic acid, or xylonic acid. See, for example, Chenand Lee, 1997, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is polyketide.

Recovery.

The fermentation product(s) can be optionally recovered from thefermentation medium using any method known in the art including, but notlimited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

Signal Peptide

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to23 of SEQ ID NO: 2 or comprising or consisting of amino acids 1 to 23 ofSEQ ID NO: 6. The polynucleotide may further comprise a gene encoding aprotein, which is operably linked to the signal peptide. The protein ispreferably foreign to the signal peptide. In one aspect, thepolynucleotide encoding the signal peptide is nucleotides 1 to 156 ofSEQ ID NO: 1. In another aspect, the polynucleotide encoding the signalpeptide is nucleotides 1 to 150 of SEQ ID NO: 5.

The present invention also relates to nucleic acid constructs,expression vectors and recombinant host cells comprising suchpolynucleotides.

The present invention also relates to methods of producing a protein,comprising (a) cultivating a recombinant host cell comprising suchpolynucleotide; and optionally (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andpolypeptides. The term “protein” also encompasses two or morepolypeptides combined to form the encoded product. The proteins alsoinclude hybrid polypeptides and fused polypeptides.

Preferably, the protein is a hormone, enzyme, receptor or portionthereof, antibody or portion thereof, or reporter. For example, theprotein may be a hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase, e.g., an alpha-galactosidase, alpha-glucosidase,aminopeptidase, amylase, beta-galactosidase, beta-glucosidase,beta-xylosidase, carbohydrase, carboxypeptidase, catalase,cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, endoglucanase, esterase,glucoamylase, invertase, laccase, lipase, mannosidase, mutanase,oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Strains

Rasamsonia byssochlamydoides strains CBS 413.71 and CBS 150.75 were usedas the source of GH10 polypeptides having xylanase activity. Aspergillusoryzae strain MT3568 was used for expression of the Rasamsoniabyssochlamydoides genes encoding the polypeptides having xylanaseactivity. A. oryzae MT3568 is an amdS (acetamidase) disrupted genederivative of Aspergillus oryzae JaL355 (WO 02/40694) in which pyrGauxotrophy was restored by disrupting the A. oryzae acetamidase (amdS)gene.

Media and Solutions

COVE sucrose plates were composed of 342 g of sucrose, 20 g of agarpowder, 20 ml of COVE salt solution, and deionized water to 1 liter. Themedium was sterilized by autoclaving at 15 psi for 15 minutes(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). Themedium was cooled to 60° C. and added 10 mM acetamide, 15 mM CsCl,Triton X-100 (50 μl/500 ml).

COVE salt solution was composed of 26 g of MgSO₄.7H₂O, 26 g of KCL, 26 gof KH₂PO₄, 50 ml of COVE trace metals solution, and deionized water to 1liter.

COVE trace metals solution was composed of 0.04 g of Na₂B₄O₇.10H₂O, 0.4g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g of MnSO₄.H₂O, 0.8 g ofNa₂MoO₄.2H₂O, 10 g of ZnSO₄.7H₂O, and deionized water to 1 liter.

Dap-4C medium was composed of 20 g of dextrose, 10 g of maltose, 11 g ofMgSO₄.7H₂O, 1 g of KH₂PO₄, 2 g of citric acid, 5.2 g of K₃PO₄.H₂O, 0.5 gof yeast extract (Difco), 1 ml of antifoam, 0.5 ml of KU6 trace metalssolution, 2.5 g of CaCO₃, and deionized water to 1 liter. The medium wassterilized by autoclaving at 15 psi for 15 minutes (BacteriologicalAnalytical Manual, 8th Edition, Revision A, 1998). Before use, 3.5 ml ofsterile 50% (NH₄)₂HPO₄ and 5 ml of sterile 20% lactic acid were addedper 150 ml of Dap-4C medium.

KU6 trace metals solution was composed of 0.13 g of NiCl₂, 2.5 g ofCuSO₄.5H₂O, 13.9 g of FeSO₄.7H₂O, 8.45 g MnSO₄—H₂O, 6.8 g ZnCl₂, 3 g ofcitric acid, and deionized water to 1 liter.

LB plates were composed of 10 g of Bacto-Tryptone, 5 g of yeast extract,10 g of sodium chloride, 15 g of Bacto-agar, and deionized water to 1liter. The medium was sterilized by autoclaving at 15 psi for 15 minutes(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998).

PDA agar plates were composed of potato infusion made by boiling 300 gof sliced potatoes (washed but unpeeled) in water for 30 minutes andthen decanting or straining the broth through cheesecloth. Distilledwater was then added until the total volume of the suspension was oneliter, followed by 20 g of dextrose and 20 g of agar powder. The mediumwas sterilized by autoclaving at 15 psi for 15 minutes (BacteriologicalAnalytical Manual, 8th Edition, Revision A, 1998).

YP+2% glucose medium was composed of 1% yeast extract, 2% peptone, and2% glucose in deionized water.

Example 1 Source of DNA Sequence Information for Rasamsoniabyssochlamydoides CBS 413.71

Genomic sequence information was generated by Illumina DNA sequencing atFasteris in Plan-les-Ouates, Switzerland from genomic DNA isolated fromRasamsonia byssochlamydoides CBS 413.71. A preliminary assembly of thegenome was analyzed using GeneMark v2.3c (Georgia Tech's Center forBioinformatics and Computational Genomics, Atlanta, Ga., USA). Genemodels constructed by the software were used as a starting point fordetecting GH10 homologs in the genome. More precise gene models wereconstructed manually using multiple known GH10 protein sequences as aguide.

Example 2 Rasamsonia byssochlamydoides CBS 413.71 and CBS 150.75 GenomicDNA Extraction

To generate genomic DNA for PCR amplification, Rasamsoniabyssochlamydoides CBS 413.71 was propagated on PDA agar plates by growthat 26° C. for 7 days. Spores harvested from the PDA plates were used toinoculate 25 ml of YP+2% glucose medium in a baffled shake flask andincubated at 26° C. for 72 hours with agitation at 85 rpm.

To generate genomic DNA for PCR amplification, Rasamsoniabyssochlamydoides CBS 150.75 was propagated on PDA agar plates by growthat 26° C. for 7 days. 50 mg of the fungal material was harvested with ascalpel by scraping off the surface of the PDA agar plate.

Genomic DNA from Rasamsonia byssochlamydoides CBS 413.71 was isolatedaccording to a modified DNEASY® Plant Maxi Kit protocol (Qiagen Danmark,Copenhagen, Denmark). The fungal material from the above culture washarvested by centrifugation at 14,000×g for 2 minutes. The supernatantwas removed and the pellet (0.5 g) was frozen in liquid nitrogen withquartz sand and ground to a fine powder in a pre-chilled mortar. Thepowder was transferred to a 15 ml centrifuge tube and 5 ml of AP1 buffer(Qiagen Danmark, Copenhagen, Denmark) preheated to 65° C. and 10 μl ofRNase A stock solution (100 mg/ml) were added followed by vigorousvortexing. After incubation for 10 minutes at 65° C. with regularinverting of the tube, 1.8 ml of AP2 buffer (Qiagen Danmark, Copenhagen,Denmark) were added to the lysate by gentle mixing followed byincubation on ice for 10 minutes. The lysate was then centrifuged at3000×g for 5 minutes at room temperature and the supernatant wasdecanted into a QIASHREDDER™ Maxi Spin Column (Qiagen Danmark,Copenhagen, Denmark), placed in a 50 ml collection tube, and centrifugedat 3000×g for 5 minutes at room temperature. The flow-through wastransferred into a new 50 ml tube and 1.5 volume of AP3/E buffer (QiagenDanmark, Copenhagen, Denmark) was added followed by vortexing. Fifteenml of the sample were transferred to a DNEASY® Maxi Spin Column placedin a 50 ml collection tube and centrifuged at 3000×g for 5 minutes atroom temperature. The flow-through was discarded and 12 ml of AW buffer(Qiagen Danmark, Copenhagen, Denmark) were added to the DNEASY® MaxiSpin Column placed in a 50 ml collection tube and centrifuged at 3000×gfor 10 minutes at room temperature. After discarding the flow-through,centrifugation was repeated to dispose of the remaining alcohol. TheDNEASY® Maxi Spin Column was transferred to a new 50 ml tube and 0.5 mlof AE buffer (Qiagen Danmark, Copenhagen, Denmark) preheated to 70° C.was added. After incubation for 5 minutes at room temperature, thesample was eluted by centrifugation at 3000×g for 5 minutes at roomtemperature. Elution was repeated with an additional 0.5 ml of AE bufferand the eluates were combined. The concentration of the harvested DNAwas measured at 260 nm using a UV spectrophotometer.

Genomic DNA isolation from Rasamsonia byssochlamydoides CBS 150.75 wascarried out using the Maxwell® 16 Instrument according to the Maxwell®16 DNA Purification Kits protocol (TH. Geyer Danmark Aps, Roskilde,Denmark). The genomic DNA was elution with 300 μl elution buffer. Theconcentration of the harvested DNA was measured at 260 nm using a UVspectrophotometer.

Example 3 Construction of an Aspergillus oryzae Expression VectorContaining Rasamsonia byssochlamydoides Strain CBS413.71 GenomicSequence Encoding a Family GH10 Polypeptide Having Xylanase Activity

Two synthetic oligonucleotide primers, shown below, were designed to PCRamplify the Rasamsonia byssochlamydoides CBS 413.71 P24GTR gene (SEQ IDNO: 1) and the Rasamsonia byssochlamydoides CBS 150.75 P34RRZ gene (SEQID NO: 5) from the genomic DNA prepared in Example 2. An IN-FUSION™Cloning Kit (BD Biosciences, Palo Alto, Calif., USA) was used to clonethe fragment directly into the expression vector pDau109 (WO2005/042735).

Primer F-P24GTR (SEQ ID NO: 3)5′-ACACAACTGGGGATCCACCATGATGGTTCGCAACCTTCCAGTC-3′ Primer R-P24GTR (SEQID NO: 4) 5′-CCCTCTAGATCTCGAG TCACAGACACTGCGAGTAATATGGATT GA-3′Bold letters represent gene sequence. The underlined sequence ishomologous to the insertion sites of pDau109.

A PHUSION® High-Fidelity PCR Kit (Finnzymes Oy, Espoo, Finland) was usedfor the PCR amplification. The PCR was composed of 5 μl of 5×HF buffer(Finnzymes Oy, Espoo, Finland), 0.5 μl of dNTPs (10 mM), 0.5 μl ofPHUSION® DNA polymerase (0.2 units/μl) (Finnzymes Oy, Espoo, Finland), 2μl of primer F-P24GTR (2.5 μM), 2 μl of primer R-P24GTR (2.5 μM), 0.5 μlof Rasamsonia byssochlamydoides genomic DNA (100 ng/μl), and 14.5 μl ofdeionized water in a total volume of 25 μl. The PCR was performed usinga PTC-200 DNA engine (MJ Research, Waltham, Mass., USA) programmed for 1cycle at 95° C. for 2 minutes; 35 cycles each at 98° C. for 10 seconds,60° C. for 30 seconds, and 72° C. for 2.5 minutes; and 1 cycle at 72° C.for 10 minutes. The sample was then held at 12° C. until removed fromthe PCR machine.

The PCR product was isolated by 1.0% agarose gel electrophoresis using40 mM Tris base, 20 mM sodium acetate, 1 mM disodium EDTA (TAE) bufferwhere a 1575 bp product band was excised from the gel and purified usingan ILLUSTRA™ GFX® PCR DNA and Gel Band Purification Kit (GE HealthcareLife Sciences, Brondby, Denmark) according to the manufacturer'sinstructions. The fragment was then cloned into Bam HI and Xho Idigested pDau109 using an IN-FUSION™ Cloning Kit according to themanufacturer's instructions resulting in plasmids pP24GTR and pP34RRZ.Cloning of the P24GTR and P34RRZ genes into Bam HI-Xho I digestedpDau109 resulted in the transcription of the Rasamsoniabyssochlamydoides P24GTR or P34RRZ gene under the control of a NA2-tpipromoter. The NA2-tpi promoter is a modified promoter from the geneencoding the Aspergillus niger neutral alpha-amylase in which theuntranslated leader has been replaced by an untranslated leader from thegene encoding the Aspergillus nidulans triose phosphate isomerase.

Plasmids pP24GTR and pP34RRZ was transformed into One Shot® TOP10F″Chemically Competent E. coli cells (Invitrogen, Carlsbad, Calif., USA)according to the manufacturer's protocol and plated onto LB platessupplemented with 0.1 mg of ampicillin per ml. After incubating at 37°C. overnight, colonies were observed growing under selection on theplates. Two colonies transformed with the P24GTR GH10 construct werecultivated in LB medium supplemented with 0.1 mg of ampicillin per mland plasmid was isolated with a QIAPREP® Spin Miniprep Kit (QIAGEN Inc.,Valencia, Calif., USA) according to the manufacturer's protocol.

Isolated plasmids were sequenced with vector primers and P24GTR genespecific primers in order to determine a representative plasmidexpression clone that was free of PCR errors.

Example 4 Characterization of the Rasamsonia byssochlamydoides GenomicSequences Encoding GH10 Polypeptides Having Xylanase Activity

DNA sequencing of the Rasamsonia byssochlamydoides CBS 413.71 P24GTR andasamsonia byssochlamydoides CBS 150.75 P34RRZ GH10 genomic clones wasperformed using an Applied Biosystems Model 3700 Automated DNA Sequencerand version 3.1 BIG-DYE™ terminator chemistry (Applied Biosystems, Inc.,Foster City, Calif., USA) and primer walking strategy. Nucleotidesequence data were scrutinized for quality and all sequences werecompared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA).

The nucleotide sequence and deduced amino acid sequence of theRasamsonia byssochlamydoides P24GTR xylanase gene are shown in SEQ IDNO: 1 and SEQ ID NO: 2, respectively. The coding sequence is 1540 byincluding the stop codon and is interrupted by four introns of 87 bp(nucleotides 63-149), 88 bp (nucleotides 304-391), 60 bp (nucleotides525-584), and 84 bp (nucleotides 699-782). The encoded predicted proteinis 406 amino acids. Using the SignalP program (Nielsen et al., 1997,Protein Engineering 10: 1-6), a signal peptide of 23 residues waspredicted. By sequence similarity, the xylanase was determined tocomprise a carbohydrate binding module (nucleotides 1436-1537). Thepredicted mature protein contains 383 amino acids with a predictedmolecular mass of 41 kDa and an isoelectric pH of 4.2.

The nucleotide sequence and deduced amino acid sequence of theRasamsonia byssochlamydoides P34RRZ xylanase gene are shown in SEQ IDNO: 5 and SEQ ID NO: 6, respectively. The coding sequence is 1590 byincluding the stop codon and is interrupted by four introns of 81 bp(nucleotides 63-143), 103 bp (nucleotides 298-400), 67 bp (nucleotides534-600), and 118 bp (nucleotides 715-832). The encoded predictedprotein is 406 amino acids. Using the SignalP program (Nielsen et al.,1997, Protein Engineering 10: 1-6), a signal peptide of 23 residues waspredicted. By sequence similarity, the xylanase was determined tocomprise a carbohydrate binding module (nucleotides 1480-1587). Thepredicted mature protein contains 383 amino acids with a predictedmolecular mass of 41 kDa and an isoelectric pH of 4.1.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman and Wunsch algorithm (Needleman andWunsch, 1970, J. Mol. Biol. 48: 443-453) with a gap open penalty of 10,a gap extension penalty of 0.5, and the EBLOSUM62 matrix. The alignmentshowed that the deduced amino acid sequences of the Rasamsoniabyssochlamydoides genes encoding the P24GTR and P34RRZ GH10 xylanaseeach share 85% identity (excluding gaps) to the deduced amino acidsequence of a predicted GH10 family protein from Rasamsonia emersonii(GENESEQP:AAU99346) with endo xylanase activity.

Example 5 Expression and Purification of the Rasamsoniabyssochlamydoides GH10 Xylanase P24GTR

The expression plasmids pP24GTR and pP34RRZ were each transformedindividually into protoplasts of Aspergillus oryzae MT3568 preparedaccording to the method of European Patent No. 0238023, pages 14-15. Thetransformation was performed according to the procedure described in WO2005/042735. Aspergillus oryzae MT3568 is an amdS (acetamidase) genedisrupted derivative of JaL355 (WO 02/40694) in which pyrG auxotrophywas restored in the process of knocking out the A, oryzae amdS gene.

Transformants were purified on COVE sucrose plates through singleconidia prior to sporulating them on PDA plates. Production of theRasamsonia byssochlamydoides GH10 polypeptide by the transformants wasanalyzed from culture supernatants of 1 ml 96 deep well stationarycultivations at 30° C. in YP+2% glucose medium. Expression was verifiedby SDS-PAGE using an E-Page 8% SDS-PAGE 48 well gel (Invitrogen,Carlsbad, Calif., USA) and Coomassie blue staining. One transformantresulting from transformation with pP24GTR was selected for further workand designated Aspergillus oryzae 69.2. One transformant resulting fromtransformation with pP34RRZ was selected for further work and designatedAspergillus oryzae 46.4.

For larger scale production, spores of Aspergillus oryzae strains 69.2and 46.4 were spread onto a PDA plate and incubated for five days at 37°C. The confluent spore plates were washed twice with 5 ml of 0.01%TWEEN® 20 to maximize the number of spores collected. The sporesuspensions of each strain were then used to inoculate twenty-five 500ml flasks each containing 100 ml of Dap-4C medium. The culture wasincubated at 30° C. with constant shaking at 100 rpm. At day fourpost-inoculation, the culture broth was collected by filtration througha bottle top MF75 Supor MachV 0.2 μm PES filter (Thermo FisherScientific, Roskilde, Denmark). SDS-PAGE analysis of fresh culture brothfrom transformants 69.2 and 46.4 each produced a band at approximately42 kDa. The identity of these prominent bands as the Rasamsoniabyssochlamydoides GH10 polypeptides was verified by peptide sequencing.

The filtrated broth from the A. oryzae strain 69.2 cultures was adjustedto pH 7.0 and filtrated using a 0.22 μm PES filter (Nalge NuncInternational, Nalgene labware cat#595-4520). Ammonium sulphate wasadded to the filtrate to a concentration of 1.8 M. The filtrate wasloaded onto a Phenyl Sepharose™ 6 Fast Flow column (high sub) (GEHealthcare, Piscataway, N.J., USA) equilibrated with 1.8 M ammoniumsulphate, 25 mM HEPES pH 7.0. After wash with 1.0 M ammonium sulphate,the bound proteins were batch eluted with 25 mM HEPES pH 7.0. Fractionswere collected and analyzed by SDS-PAGE. The fractions were pooled andapplied to a Sephadex™ G-25 (medium) (GE Healthcare, Piscataway, N.J.,USA) column equilibrated in 25 mM HEPES pH 7.0. Fractions werecollected, pooled, and applied to a SOURCE™ 15Q (GE Healthcare,Piscataway, N.J., USA) column equilibrated in 25 mM HEPES pH 7.0 andbound proteins were eluted with a linear gradient from 0-1000 mM sodiumchloride over 20 column volumes. Fractions were collected and analyzedby SDS-PAGE.

Example 6 Alternative Method for Producing the Rasamsoniabyssochlamydoides GH10 Xylanases

Based on the nucleotide sequences identified as SEQ ID NO: 1 and SEQ IDNO: 5, synthetic genes can be obtained from a number of vendors such asGene Art (GENEART AG BioPark, Josef-Engert-Str. 11, 93053, Regensburg,Germany) or DNA 2.0 (DNA2.0, 1430 O'Brien Drive, Suite E, Menlo Park,Calif. 94025, USA). The synthetic genes can be designed to incorporateadditional DNA sequences such as restriction sites or homologousrecombination regions to facilitate cloning into an expression vector.

Using the two synthetic oligonucleotide primers F-P24GTR and F-P24GTRdescribed above, PCR can be used to amplify the full-length open readingframe from the synthetic gene of SEQ ID NO: 1 or of SEQ ID NO: 5. Thegene can then be cloned into an expression vector, e.g., as describedabove, and expressed in a host cell, e.g., Aspergillus oryzae asdescribed above.

Example 7 Pretreated Corn Cobs Hydrolysis Assay

Corn cobs were pretreated with NaOH (0.08 g/g dry weight cobs) at 120°C. for 60 minutes at 15% total dry weight solids (TS). The resultingmaterial was washed with water until it was pH 8.2, resulting in washedalkaline pretreated corn cobs (APCC). Ground Sieved Alkaline PretreatedCorn Cobs (GS-APCC) was prepared by adjusting the pH of APCC to 5.0 byaddition of 6 M HCl and water with extensive mixing, milling APCC in aCosmos ICMG 40 wet multi-utility grinder (EssEmm Corporation, TamilNadu, India), and autoclaving for 45 minutes at 121° C., with a final TSof 3.33%. The hydrolysis of GS-APCC was conducted using 2.2 ml deep-wellplates (Axygen, Union City, Calif., USA) in a total reaction volume of1.0 ml.

The hydrolysis was performed with 10 mg of GS-APCC total solids per mlof 50 mM sodium acetate (pH 4.0 to 5.5) or 50 mM Tris (pH 6.0 to 7.0)buffer containing 1 mM manganese sulfate and various protein loadings ofvarious enzyme compositions (expressed as mg protein per gram ofcellulose). Enzyme compositions were prepared and then addedsimultaneously to all wells in a volume ranging from 50 μl to 200 μl,for a final volume of 1 ml in each reaction. The plate was then sealedusing an ALPS300™ plate heat sealer (Abgene, Epsom, United Kingdom),mixed thoroughly, and incubated at a specific temperature for 72 hours.All experiments reported were performed in triplicate.

Following hydrolysis, samples were filtered using a 0.45 μm MULTISCREEN®96-well filter plate (Millipore, Bedford, Mass., USA) and filtrates wereanalyzed for sugar content as described below. When not usedimmediately, filtered aliquots were frozen at −20° C. The sugarconcentrations of samples diluted in 0.005 M H₂SO₄ were measured using a4.6×250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules,Calif., USA) by elution with 0.05% w/w benzoic acid-0.005 M H₂SO₄ at 65°C. at a flow rate of 0.6 ml per minute, and quantitation by integrationof the glucose, cellobiose, and xylose signals from refractive indexdetection (CHEMSTATION®, AGILENT® 1100 HPLC, Agilent Technologies, SantaClara, Calif., USA) calibrated by pure sugar samples. The resultantglucose equivalents were used to calculate the percentage of celluloseconversion for each reaction. The resultant xylose equivalents were usedto calculate the percentage of xylo-oligosaccharide conversion for eachreaction.

Glucose, cellobiose, and xylose were measured individually. Measuredsugar concentrations were adjusted for the appropriate dilution factor.All HPLC data processing was performed using MICROSOFT EXCEL™ software(Microsoft, Richland, Wash., USA).

The degree of xylo-oligosaccharide conversion to xylose was calculatedusing the following equation: % xylose conversion=xyloseconcentration/xylose concentration in a limit digest. In order tocalculate % conversion, a 100% conversion point was set based on acellulase control (100 mg of Trichoderma reesei cellulase supplementedwith P. emersonii GH61A polypeptide (WO 2011/041397), A. fumigatus GH10xylanase (xyn3) (WO 2006/078256), and T. emersonii GH3 beta-xylosidase(WO 2003/070956) per gram cellulose), and all values were divided bythis number and then multiplied by 100. The % relative activity for eachtemperature was calculated using the following equation: % relativeactivity=(% xylose conversion of a xylanase at a certain pH andtemperature−% xylose conversion of beta-xylosidase at that certain pHand temperature)/(% xylose conversion of the xylanase for the pH andtemperature containing the highest % xylose conversion−% xyloseconversion of beta-xylosidase for the pH and temperature containing thehighest % xylose conversion)×100.

Example 8 Preparation of Talaromyces emersonii CBS 393.64 GH3Beta-Xylosidase

A Talaromyces emersonii CBS 393.64 beta-xylosidase (GENESEQP:AZI104896)was prepared recombinantly according to Rasmussen et al., 2006,Biotechnology and Bioengineering 94: 869-876 using Aspergillus oryzaeJaL355 as a host (WO 2003/070956). The filtered broth was concentratedand desalted with 50 mM sodium acetate pH 5.0 using a tangential flowconcentrator equipped with a 10 kDa polyethersulfone membrane. Proteinconcentration was determined using a Microplate BCA™ Protein Assay Kit(Thermo Fischer Scientific, Waltham, Mass., USA) in which bovine serumalbumin was used as a protein standard.

Example 9 Preparation of Aspergillus fumigatus GH10 Xylanase (P4D6)

Aspergillus fumigatus NN055679 GH10 xylanase (xyn3) (GENESEQP:AEC74753)was prepared recombinantly according to WO 2006/078256 using Aspergillusoryzae BECh2 (WO 00/39322) as a host.

The filtered broth was desalted and buffer-exchanged into 50 mM sodiumacetate pH 5.0 using a HIPREP® 26/10 Desalting Column (GE Healthcare,Piscataway, N.J., USA) according to the manufacturer's instructions.Protein concentration was determined using a Microplate BCA™ ProteinAssay Kit with bovine serum albumin as a protein standard.

Example 10 Effect of Rasamsonia byssochlamydoides GH10 Xylanase (P24GTR)Supplemented with Talaromyces emersonii GH3 Beta-Xylosidase UsingGS-APCC at pH 4.0 to 7.0

The Rasamsonia byssochlamydoides GH10 xylanase (P24GTR) supplementedwith Talaromyces emersonii GH3 beta-xylosidase (Example 8) was evaluatedat 50° C., 55° C., 60° C., 65° C. from pH 4.0 to 7.0 using washedground-sieved alkaline pretreated corn cobs (GS-APCC) as a substrate.The Aspergillus fumigatus GH10 xylanase (P4D6) supplemented withTalaromyces emersonii GH3 beta-xylosidase was also tested forcomparison. The xylanases were added to the GS-APCC hydrolysis at 1 mgtotal protein per g cellulose supplemented with beta-xylosidase at 4.0mg total protein per g cellulose.

The assay was performed as described in Example 7. The 1 ml reactionswith GS-APCC (1% total solids) were conducted for 72 hours in 50 mMsodium acetate (pH 4.0 to 5.5) or 50 mM Tris (pH 6.0 to 7.0) buffercontaining 1 mM manganese sulfate. All reactions were performed intriplicate and involved single mixing at the beginning of hydrolysis.

The results for the hydrolysis at pH 4.0 from 50° C. to 65° C. are shownin FIG. 1. The relative activity results for Rasamsoniabyssochlamydoides GH10 xylanase are shown in Table 1 where the resultsare compared to a temperature of 65° C. and pH 4.0, and the relativeactivity results for Aspergillus fumigatus GH10 xylanase are shown inTable 2 where the results are compared to a temperature of 55° C. and pH5.5. As shown in FIG. 1, the Rasamsonia byssochlamydoides GH10 xylanasesupplemented with beta-xylosidase had significantly higher activity thanthe Aspergillus fumigatus GH10 xylanase supplemented withbeta-xylosidase at all temperatures. In addition, the Rasamsoniabyssochlamydoides GH10 xylanase supplemented with beta-xylosidase hadincreasing activity as the temperature increased from 50° C. to 65° C.while the Aspergillus fumigatus GH10 xylanase supplemented withbeta-xylosidase had decreasing activity as temperature increased from50° C. to 65° C. As shown in Table 1, the Rasamsonia byssochlamydoidesGH10 xylanase had an optimal temperature of 65° C. and an optimal pHrange (greater than 75% relative activity) from pH 4.0 to 6.0 at theoptimal temperature. In comparison, the Aspergillus fumigatus GH10xylanase had an optimal temperature of 55° C. and an optimal pH range(greater than 75% relative activity) from pH 4.5 to 6.0 at 55° C.Overall, the Rasamsonia byssochlamydoides GH10 xylanase had a higheroptimal temperature activity and activity at lower pH than theAspergillus fumigatus GH10 xylanase.

TABLE 1 pH 4.0 pH 4.5 pH 5.0 pH 5.5 pH 6.0 pH 7.0 % % % % % % RelativeRelative Relative Relative Relative Relative Sample Activity ActivityActivity Activity Activity Activity R. byssochla- 80% 79% 73% 66% 62%40% mydoides GH10 xyn (50° C.) (P24GTR) R. byssochla- 83% 81% 77% 72%68% 34% mydoides GH10 xyn (55° C.) (P24GTR) R. byssochla- 93% 87% 81%71% 75% 27% mydoides GH10 xyn (60° C.) (P24GTR) R. byssochla- 100%  95%88% 76% 83% 21% mydoides GH10 xyn (65° C.) (P24GTR)

TABLE 2 pH 4.0 pH 4.5 pH 5.0 pH 5.5 pH 6.0 pH 7.0 % % % % % % RelativeRelative Relative Relative Relative Relative Sample Activity ActivityActivity Activity Activity Activity A. fumigatus 73% 92% 96% 94% 91% 69%GH10 xyn3 (50° C.) A. fumigatus 64% 90% 96% 100%  96% 54% GH10 xyn3 (55°C.) A. fumigatus 56% 79% 98% 98% 93% 34% GH10 xyn3 (60° C.) A. fumigatus54% 70% 87% 89% 87% 20% GH10 xyn3 (65° C.)

Example 11 Specific Activity of Rasamsonia byssochlamydoides GH10Xylanase and Aspergillus fumigatus GH10 Xylanase on Birchwood Xylan atpH 5.0, 50° C.

The specific activities of the Rasamsonia byssochlamydoides GH10xylanase (P24GTR) and Aspergillus fumigatus GH10 xylanase weredetermined on birchwood xylan (Sigma Chemical Co., Inc., St. Louis, Mo.,USA). A solution of birchwood xylan at 2 g per liter was prepared in 50mM sodium acetate buffer, pH 5.0. Ten μl of xylanase was added to 190 μlof the 2 g/L of birchwood xylan at different enzyme loadings. Substratealone and enzyme alone controls were also run. The reactions wereincubated at 50° C. for 30 minutes and then stopped by adding 50 μl of0.5 M NaOH to each reaction. The reducing sugars produced weredetermined using a para-hydroxybenzoic acid hydrazide (PHBAH, Sigma, St.Louis, Mo., USA) assay adapted to a 96 well microplate format asdescribed below. Briefly, a 100 μl aliquot of an appropriately dilutedsample was placed in a 96-well conical bottomed microplate. Reactionswere initiated by adding 50 μl of 1.5% (w/v) PHBAH in 2% NaOH to eachwell. Plates were heated uncovered at 95° C. for 10 minutes. Plates wereallowed to cool to room temperature and 50 μl of Milli Q H₂O (Millipore,Bedford, Mass., USA) were added to each well. A 100 μl aliquot from eachwell was transferred to a flat bottomed 96 well plate and the absorbanceat 410 nm was measured using a SpectraMax Microplate Reader (MolecularDevices, Sunnyvale, Calif.). Glucose standards (0.1-0.0125 mg/ml dilutedwith 0.4% sodium hydroxide) were used to prepare a standard curve totranslate the obtained A_(410nm) values into glucose equivalents. Theenzyme loading versus the reducing sugars produced was plotted and thelinear range was used to calculate the specific activity of Rasamsoniabyssochlamydoides GH10 xylanase and Aspergillus fumigatus GH10 xylanase,as expressed as μmole glucose equivalent produced per minute per mgenzyme, or IU/mg. The specific activity results are shown in Table 3.

TABLE 3 Specific activity on birchwood xylan at pH 5.0, 50° C. Specificactivity on birchwood Enzyme xylan, IU/mg Rasamsonia byssochlamydoides111.6 +/− 8.6 GH10 xylanase (P24GTR) Aspergillus fumigatus   16 +/− 4.2GH10 xylanase

Example 12 Preparation of Rasamsonia emersonii GH10 Xylanase (P23CQ3)

Rasamsonia emersonii GH10 xylanase (GENESEQP:AZI05030) was preparedrecombinantly and purified according to WO 2011/057140 using Aspergillusoryzae (WO 00/39322) as a host. Protein concentration was determinedusing a Microplate BCA™ Protein Assay Kit with bovine serum albumin as aprotein standard.

Example 13 Specific Activity of Rasamsonia byssochlamydoides GH10Xylanase and Rasamsonia emersonii GH10 Xylanase on Birchwood Xylan at pH4.0, 60° C.

The specific activities of the Rasamsonia byssochlamydoides GH10xylanase (P24GTR) and Rasamsonia emersonii GH10 xylanase were determinedon birchwood xylan (Sigma Chemical Co., Inc.) as described in Example11. The solution of birchwood xylan at 2 g per liter was prepared in 50mM sodium acetate buffer, pH 4.0, and reactions were incubated at 60° C.for 30 minutes. Xylose standards (0.1-0.0125 mg/ml diluted with 0.4%sodium hydroxide) were used to prepare a standard curve to translate theobtained A_(410nm) values into xylose equivalents. The specific activityresults are shown in Table 4.

TABLE 4 Specific activity on birchwood xylan at pH 4.0, 60° C. Specificactivity on birchwood Enzyme xylan, IU/mg (pH 4.0, 60° C.) Rasamsoniabyssochlamydoides   652 +/− 107.3 GH10 xylanase (P24GTR) Rasamsoniaemersonii 317.8 +/− 58.9 GH10 xylanase (P23CQ3)

Example 14 Preparation of the Natural Variant Rasamsoniabyssochlamydoides GH10 Xylanase P34RRZ

The protein broth of Rasamsonia byssochlamydoides GH10 xylanase P34RRZwas desalted and buffer exchanged in 50 mM sodium acetate pH 5.0 usingan ECONO-PAC® 10-DG desalting column (Bio-Rad Laboratories, Inc.)according to the manufacturer's instructions. Protein concentration wasdetermined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard.

Example 15 Specific Activity of the Natural Variant Rasamsoniabyssochlamydoides GH10 Xylanase P34RRZ on Birchwood Xylan at pH 4.0, 60°C.

The specific activity of the natural variant Rasamsoniabyssochlamydoides GH10 xylanase (P34RRZ) was determined on birchwoodxylan (Sigma Chemical Co., Inc.) as described in Example 13. Thespecific activity results are shown in Table 5.

TABLE 5 Specific activity on birchwood xylan at pH 4.0, 60° C. Specificactivity on birchwood Enzyme xylan, IU/mg (pH 4.0, 60° C.) Rasamsoniabyssochlamydoides 578.2 +/− 98.7 GH10 xylanase (P34RRZ)

The present invention is further described by the following numberedparagraphs:

[1] An isolated polypeptide having xylanase activity, selected from thegroup consisting of:

(a) a polypeptide having at least 90% sequence identity to the maturepolypeptide of SEQ ID NO: 2 or SEQ ID NO: 6;

(b) a polypeptide encoded by a polynucleotide having at least 90%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the cDNA sequence thereof, or to the mature polypeptide codingsequence of SEQ ID NO: 5 or the cDNA sequence thereof;

(c) a variant of the mature polypeptide of SEQ ID NO: 2 or of the maturepolypeptide of SEQ ID NO: 4 comprising a substitution, deletion, and/orinsertion at one or more positions; and

(d) a fragment of the polypeptide of (a), (b), or (c), that has xylanaseactivity.

[2] The polypeptide of paragraph [1], having at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to themature polypeptide of SEQ ID NO: 2.

[3] The polypeptide of paragraph [1] or [2], having at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to the mature polypeptide of SEQ ID NO: 6.

[4] The polypeptide of any of paragraphs [1]-[3], which is encoded by apolynucleotide having at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 1 or the cDNA sequence thereof.

[5] The polypeptide of any of paragraphs [1]-[4], which is encoded by apolynucleotide having at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 5 or the cDNA sequence thereof.

[6] The polypeptide of any of paragraphs [1]-[5], comprising orconsisting of SEQ ID NO: 2 or the mature polypeptide thereof.

[7] The polypeptide of paragraph [6], wherein the mature polypeptide isamino acids 24 to 406 of SEQ ID NO: 2.

[8] The polypeptide of any of paragraphs [1]-[5], comprising orconsisting of SEQ ID NO: 6, or the mature polypeptide thereof.

[9] The polypeptide of paragraph [8], wherein the mature polypeptide isamino acids 24 to 406 of SEQ ID NO: 6.

[10] The polypeptide of any of paragraphs [1]-[5], which is a variant ofthe mature polypeptide of SEQ ID NO: 2, or of the mature polypeptide ofSEQ ID NO: 6, comprising a substitution, deletion, and/or insertion atone or more positions.

[11] The polypeptide of paragraph [1], which is a fragment of SEQ ID NO:2 or a fragment of SEQ ID NO: 6, wherein the fragment has xylanaseactivity.

[12] The polypeptide of any of paragraphs [1]-[11], which is encoded bythe polynucleotide contained in Rasamsonia byssochiamydoides CBS 413.71.

[13] The polypeptide of any of paragraphs [1]-[12], which has at least10%, e.g., at least 15% and at least 20%, more xylanase activity at pH4.0 and 60° C. or 65° C. than at pH 4.0 and 50° C.

[14] The polypeptide of any of paragraphs [1]-[13], which has at least10%, e.g., at least 15% and at least 20%, more xylanase activity at pH4.0 and 50° C., 55° C., 60° C., or 65° C. than at pH 6.0 and 50° C., 55°C., 60° C., or 65° C., respectively.

[15] An isolated polypeptide having xylanase activity, which has atleast 10%, e.g., at least 15% and at least 20%, more xylanase activityat pH 4.0 and 60° C. or 65° C. than at pH 4.0 and 50° C.

[16] An isolated polypeptide having xylanase activity, which has atleast 10%, e.g., at least 15% and at least 20%, more xylanase activityat pH 4.0 and 50° C., 55° C., 60° C., or 65° C. than at pH 6.0 and 50°C., 55° C., 60° C., or 65° C., respectively.

[17] An isolated polypeptide comprising a catalytic domain selected fromthe group consisting of:

(a) a catalytic domain having at least 60% sequence identity to aminoacids 24 to 340 of SEQ ID NO: 2, or having at least 60% sequenceidentity to amino acids 24 to 341 of SEQ ID NO: 6;

(b) a catalytic domain encoded by a polynucleotide having at least 90%sequence identity to nucleotides 157 to 1339 of SEQ ID NO: 1 or the cDNAsequence thereof, or having at least 90% sequence identity tonucleotides 151 to 1387 of SEQ ID NO: 5 or the cDNA sequence thereof;

(c) a variant of amino acids 24 to 340 of SEQ ID NO: 2, or of aminoacids 24 to 341 of SEQ ID NO: 6, comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions; and

(d) a fragment of the catalytic domain of (a), (b), or (c) that hasxylanase activity.

[18] The polypeptide of paragraph [17], further comprising acarbohydrate binding module.

[19] An isolated polypeptide comprising a carbohydrate binding moduleoperably linked to a catalytic domain, wherein the binding domain isselected from the group consisting of:

(a) a carbohydrate binding module having at least 90% sequence identityto amino acids 373 to 406 of SEQ ID NO: 2 or having at least 90%sequence identity to amino acids 371 to 406 of SEQ ID NO: 6;

(b) a carbohydrate binding module encoded by a polynucleotide having atleast 90% sequence identity to nucleotides 1436 to 1537 of SEQ ID NO: 1or the cDNA sequence thereof, or by a polynucleotide having at least 90%sequence identity to nucleotides 1480 to 1587 of SEQ ID NO: 5 or thecDNA sequence thereof;

(c) a variant of amino acids 373 to 406 of SEQ ID NO: 2, or of aminoacids 3731 to 406 of SEQ ID NO: 6, comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions; and

(d) a fragment of the carbohydrate binding module of (a), (b), or (c)that has binding activity.

[20] The polypeptide of paragraph [19], wherein the catalytic domain isobtained from a hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase, e.g., an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase,lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase, xylanase, or beta-xylosidase.

[21] A composition comprising the polypeptide of any of paragraphs[1]-[20].

[22] An isolated polynucleotide encoding the polypeptide of any ofparagraphs [1]-[20].

[23] A nucleic acid construct or expression vector comprising thepolynucleotide of paragraph [22] operably linked to one or more controlsequences that direct the production of the polypeptide in an expressionhost.

[24] A recombinant host cell comprising the polynucleotide of paragraph[22] operably linked to one or more control sequences that direct theproduction of the polypeptide.

[25] A method of producing the polypeptide of any of paragraphs[1]-[20], comprising: cultivating a cell, which in its wild-type formproduces the polypeptide, under conditions conducive for production ofthe polypeptide.

[26] The method of paragraph [25], further comprising recovering thepolypeptide.

[27] A method of producing a polypeptide having xylanase activity,comprising: cultivating the host cell of paragraph [24] under conditionsconducive for production of the polypeptide.

[28] The method of paragraph [27], further comprising recovering thepolypeptide.

[29] A transgenic plant, plant part or plant cell transformed with apolynucleotide encoding the polypeptide of any of paragraphs [1]-[20].

[30] A method of producing a polypeptide having xylanase activity,comprising: cultivating the transgenic plant or plant cell of paragraph[29] under conditions conducive for production of the polypeptide.

[31] The method of paragraph [30], further comprising recovering thepolypeptide.

[32] A method of producing a mutant of a parent cell, comprisinginactivating a polynucleotide encoding the polypeptide of any ofparagraphs [1]-[20], which results in the mutant producing less of thepolypeptide than the parent cell.

[33] A mutant cell produced by the method of paragraph [32].

[34] The mutant cell of paragraph [33], further comprising a geneencoding a native or heterologous protein.

[35] A method of producing a protein, comprising: cultivating the mutantcell of paragraph [33] or [34] under conditions conducive for productionof the protein.

[36] The method of paragraph [35], further comprising recovering theprotein.

[37] A double-stranded inhibitory RNA (dsRNA) molecule comprising asubsequence of the polynucleotide of paragraph [22], wherein optionallythe dsRNA is an siRNA or an miRNA molecule.

[38] The double-stranded inhibitory RNA (dsRNA) molecule of paragraph[37], which is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or moreduplex nucleotides in length.

[39] A method of inhibiting the expression of a polypeptide havingxylanase activity in a cell, comprising administering to the cell orexpressing in the cell the double-stranded inhibitory RNA (dsRNA)molecule of paragraph [37] or [38].

[40] A cell produced by the method of paragraph [39].

[41] The cell of paragraph [40], further comprising a gene encoding anative or heterologous protein.

[42] A method of producing a protein, comprising: cultivating the cellof paragraph [40] or [41] under conditions conducive for production ofthe protein.

[43] The method of paragraph [42], further comprising recovering theprotein.

[44] An isolated polynucleotide encoding a signal peptide comprising orconsisting of amino acids 1 to 23 of SEQ ID NO: 2 or of amino acids 1 to23 of SEQ ID NO: 6.

[45] A nucleic acid construct or expression vector comprising a geneencoding a protein operably linked to the polynucleotide of paragraph[44], wherein the gene is foreign to the polynucleotide encoding thesignal peptide.

[46] A recombinant host cell comprising a gene encoding a proteinoperably linked to the polynucleotide of paragraph [44], wherein thegene is foreign to the polynucleotide encoding the signal peptide.

[47] A method of producing a protein, comprising: cultivating arecombinant host cell comprising a gene encoding a protein operablylinked to the polynucleotide of paragraph [40], wherein the gene isforeign to the polynucleotide encoding the signal peptide, underconditions conducive for production of the protein.

[48] The method of paragraph [47], further comprising recovering theprotein.

[49] A process for degrading a cellulosic or xylan-containing material,comprising: treating the cellulosic or xylan-containing material with anenzyme composition in the presence of the polypeptide having xylanaseactivity of any of paragraphs [1]-[20].

[50] The process of paragraph [49], wherein the cellulosic orxylan-containing material is pretreated.

[51] The process of paragraph [49] or [50], wherein the enzymecomposition comprises one or more enzymes selected from the groupconsisting of a cellulase, a polypeptide having cellulolytic enhancingactivity, a hemicellulase, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.

[52] The process of paragraph [51], wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[53] The process of paragraph [51], wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[54] The process of any of paragraphs [49]-[53], further comprisingrecovering the degraded cellulosic or xylan-containing material.

[55] The process of paragraph [54], wherein the degraded cellulosic orxylan-containing material is a sugar.

[56] The process of paragraph [55], wherein the sugar is selected fromthe group consisting of glucose, xylose, mannose, galactose, andarabinose.

[57] A process for producing a fermentation product, comprising:

(a) saccharifying a cellulosic or xylan-containing material with anenzyme composition in the presence of the polypeptide having xylanaseactivity of any of paragraphs [1-16];

(b) fermenting the saccharified cellulosic or xylan-containing materialwith one or more fermenting microorganisms to produce the fermentationproduct; and

(c) recovering the fermentation product from the fermentation.

[58] The process of paragraph [57], wherein the cellulosic orxylan-containing material is pretreated.

[59] The process of paragraph [57] or [58], wherein the enzymecomposition comprises the enzyme composition comprises one or moreenzymes selected from the group consisting of a cellulase, a polypeptidehaving cellulolytic enhancing activity, a hemicellulase, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.

[60] The process of paragraph [59], wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[61] The process of paragraph [59] or [60], wherein the hemicellulase isone or more enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[62] The process of any of paragraphs [57]-[61], wherein steps (a) and(b) are performed simultaneously in a simultaneous saccharification andfermentation.

[63] The process of any of paragraphs [57]-[62], wherein thefermentation product is an alcohol, an alkane, a cycloalkane, an alkene,an amino acid, a gas, isoprene, a ketone, an organic acid, orpolyketide.

[64] A process of fermenting a cellulosic or xylan-containing material,comprising: fermenting the cellulosic or xylan-containing material withone or more fermenting microorganisms, wherein the cellulosic orxylan-containing material is saccharified with an enzyme composition inthe presence of the polypeptide having xylanase activity of any ofparagraphs [1]-[20].

[65] The process of paragraph [64], wherein the fermenting of thecellulosic or xylan-containing material produces a fermentation product.

[66] The process of paragraph [65], further comprising recovering thefermentation product from the fermentation.

[67] The process of any of paragraphs [64]-[66], wherein the cellulosicor xylan-containing material is pretreated before saccharification.

[68] The process of any of paragraphs [64]-[67], wherein the enzymecomposition comprises one or more enzymes selected from the groupconsisting of a cellulase, a polypeptide having cellulolytic enhancingactivity, a hemicellulase, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.

[69] The process of paragraph [68], wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[70] The process of paragraph [68], wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[71] The process of any of paragraphs [65]-[70], wherein thefermentation product is an alcohol, an alkane, a cycloalkane, an alkene,an amino acid, a gas, isoprene, a ketone, an organic acid, orpolyketide.

[72] A whole broth formulation or cell culture composition comprisingthe polypeptide of any of paragraphs [1]-[20].

[73] An enzyme composition comprising the polypeptide having xylanaseactivity of any of paragraphs [1]-[20] and one or more enzymes selectedfrom the group consisting of a cellulase, a polypeptide havingcellulolytic enhancing activity, a hemicellulase, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.

[74] The enzyme composition of paragraph [73], wherein the cellulase isone or more enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[75] The enzyme composition of paragraph [73] or [74], wherein thehemicellulase is one or more enzymes selected from the group consistingof a xylanase, an acetylxylan esterase, a feruloyl esterase, anarabinofuranosidase, a xylosidase, and a glucuronidase.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1. An isolated polypeptide having xylanase activity, selected from thegroup consisting of: (a) a polypeptide having at least 90% sequenceidentity to amino acids 24 to 406 of SEQ ID NO: 2 or at least 90%sequence identity to amino acids 24 to 406 of SEQ ID NO: 6; (b) avariant of amino acids 24 to 406 of SEQ ID NO: 2, or of amino acids 24to 406 of SEQ ID No: 6, comprising a substitution, deletion, and/orinsertion at one or more positions; and (c) a fragment of thepolypeptide of (a), or (b), that has xylanase activity.
 2. (canceled) 3.(canceled)
 4. (canceled)
 5. (canceled)
 6. (canceled)
 7. (canceled)
 8. Anenzyme composition comprising the polypeptide having xylanase activityof claim
 1. 9. An enzyme composition comprising the polypeptide havingxylanase activity of claim 1 and one or more enzymes selected from thegroup consisting of a cellulase, a polypeptide having cellulolyticenhancing activity, a hemicellulase, an esterase, an expansin, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.
 10. (canceled)
 11. (canceled)
 12. A process fordegrading a cellulosic or xylan-containing material, comprising:treating the cellulosic or xylan-containing material with the enzymecomposition of claim
 8. 13. The process of claim 12, wherein thecellulosic or xylan-containing material is pretreated.
 14. The processof claim 12, further comprising recovering the degraded cellulosic orxylan-containing material.
 15. The process of claim 14, wherein thedegraded cellulosic or xylan-containing material is a sugar.
 16. Aprocess for producing a fermentation product, comprising: (a)saccharifying a cellulosic or xylan-containing material with the enzymecomposition of claim 8; (b) fermenting the saccharified cellulosic orxylan-containing material with one or more fermenting microorganisms toproduce the fermentation product; and (c) recovering the fermentationproduct from the fermentation.
 17. The process of claim 16, wherein thecellulosic or xylan-containing material is pretreated.
 18. The processof claim 17, wherein steps (a) and (b) are performed simultaneously in asimultaneous saccharification and fermentation.
 19. The process of claim16, wherein the fermentation product is an alcohol, an alkane, acycloalkane, an alkene, an amino acid, a gas, isoprene, a ketone, anorganic acid, or polyketide.
 20. A whole broth formulation or cellculture composition comprising the polypeptide of claim
 1. 21. Anisolated polynucleotide encoding the polypeptide of claim
 1. 22. Anucleic acid construct or expression vector comprising thepolynucleotide of claim 21 operably linked to one or more heterologouscontrol sequences that direct the production of the polypeptide in anexpression host.
 23. A recombinant host cell transformed with thenucleic acid construct or expression vector of claim
 22. 24. A method ofproducing a polypeptide having xylanase activity, comprising:cultivating the recombinant host cell of claim 23 under conditionsconducive for production of the polypeptide.
 25. The method of claim 24,further comprising recovering the polypeptide.
 26. A transgenic plant,plant part or plant cell transformed with the nucleic acid construct orexpression vector of claim
 22. 27. A method of producing a polypeptidehaving xylanase activity, comprising: cultivating the transgenic plantor plant cell of claim 26 under conditions conducive for production ofthe polypeptide.
 28. The method of claim 27, further comprisingrecovering the polypeptide.